Full text of "USPTO Patents Application 10551504"

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WORLD INTELLECTUAL PROPERTY ORGANIZATION

International Bureau

PCT

INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)

(51) International Patent Classification 6 :

C12N 15/09, 15/17, 15/63, C07H 21/04,
C07K 1/14, 14/52, 16/24, C12P 1/20,
21/08

(11) International Publication Number:
(43) International Publication Date:

WO 97/01633

16 January 1997 (16.01.97)

(21) International Application Number:

(22) International Filing Date:

PCT/US96/ 10895

25 June 1996 (25.06.96)

(30) Priority Data:

08/496,632
08/548,368

29 June 1995 (29.06.95) US
1 November 1 995 (01 . 1 1 .95) US

(81) Designated States: AU, CA, IL, JP, KR, MX, NO, NZ,
European patent (AT, BE, CH, DE, DK, ES, FI, FR, GB,
GR, IE, IT, LU, MC, NL, PT, SE).

Published

With international search report.

(71) Applicant: IMMUNEX CORPORATION [US/US]; 51 Univer-

sity Street, Seattle, WA 98101 (US).

(72) Inventors: WILEY, Steven, R.; 95 1 North 42nd Street, Seattle,

WA 98013 (US). GOODWIN, Raymond, G.; 3322 8th
Avenue West, Seattle, WA 98119 (US).

(74) Agent: ANDERSON, Kathryn, A.; Immunex Corporation, 51
University Street, Seattle, WA 98101 (US).

(54) Title: CYTOKINE THAT INDUCES APOPTOSIS

(57) Abstract

A novel cytokine designated TRAIL induces apoptosis of certain target cells, including cancer cells and virally infected cells. Isolated
DNA sequences encoding TRAIL are disclosed, along with expression vectors and transformed host cells useful in producing TRAIL
polypeptides. Antibodies that specifically bind TRAIL are provided as well.

FOR THE PURPOSES OF INFORMATION ONLY

Codes used to identify States party to the PCT on the front pages of pamphlets publishing international
applications under the PCT.

Armenia

Austria

Australia

Barbados

Belgium

Burkina Faso

Bulgaria

Benin

Brazil

Belarus

Canada

Central African Republic

Congo

Switzerland

Cdte d'lvoire

Cameroon

China

Czechoslovakia

Czech Republic

Germany

Denmark

Estonia

Spain

Finland

France

Gabon

United Kingdom

Georgia

Guinea

Greece

Hungary

Ireland

Italy

Japan

Kenya

Kyrgystan

Democratic People's Republic

of Korea

Republic of Korea

Kazakhstan

Liechtenstein

Sri Lanka

Liberia

Lithuania

Luxembourg

Latvia

Monaco

Republic of Moldova

Madagascar

Mali

Mongolia

Mauritania

Malawi

Mexico

Niger

Netherlands

Norway

New Zealand

Poland

Portugal

Romania

Russian Federation

Sudan

Sweden

Singapore

Slovenia

Slovakia

Senegal

Swaziland

Chad

Togo

Tajikistan

Trinidad and Tobago

Ukraine

Uganda

United States of America

Uzbekistan

Viet Nam

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TITLE

CYTOKINE THAT INDUCES APOPTOSIS

INVENTION

10 The programmed cell death known as apoptosis is distinct from cell death due to

necrosis, Apoptosis occurs in embryogenesis, metamorphosis, endocrine-dependent
tissue atrophy, normal tissue turnover, and death of immune thymocytes (induced
through their antigen-receptor complex or by glucocorticoids) (Itoh et al., Cell 66:233,
1991). During maturation of T-cells in the thymus, T-cells that recognize self-antigens

15 are destroyed through the apoptotic process, whereas others are positively selected.
The possibility that some T-cells recognizing certain self epitopes (e.g., inefficiently
processed and presented antigenic determinants of a given self protein) escape this
elimination process and subsequently play a role in autoimmune diseases has been
suggested (Gammon et al., Immunology Today 12:193, 1991).

20 A cell surface antigen known as Fas has been reported to mediate apoptosis and

is believed to play a role in clonal deletion of self-reactive T-cells (Itoh et al., Cell
66:233, 1991; Watanabe-Fukunage et ^Nature 356:314, 1992). Cross-linking a
specific monoclonal antibody to Fas has been reported to induce various cell lines to
undergo apoptosis (Yonehara et al., /. Exp. Med., 169:1747, 1989; Trauth et al.,

25 Science, 245:301, 1989). However, under certain conditions, binding of a specific
monoclonal antibody to Fas can have a costimulatory effect on freshly isolated T cells
(Alderson et al., /. Exp. Med. 178:2231, 1993).

DNAs encoding a rat Fas ligand (Suda et al., Cell 75 : 1 169, 1993) and a human
Fas ligand (Takahashi et al., International Immunology 6:1567, 1994) have been

30 isolated. Binding of the Fas ligand to cells expressing Fas antigen has been

demonstrated to induce apoptosis (Suda et al., supra, and Takahashi et al., supra).

Investigation into the existence and identity of other molecule(s) that play a role
in apoptosis is desirable. Identifying such molecules would provide an additional
means of regulating apoptosis, as well as providing further insight into the development

35 of self-tolerance by the immune system and the etiology of autoimmune diseases.

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SUMMARY OF THE INVENTION

The present invention provides a novel cytokine protein, as well as isolated
DNA encoding the cytokine and expression vectors comprising the isolated DNA.
Properties of the novel cytokine, which is a member of the tumor necrosis factor (TNF)
5 family of ligands, include the ability to induce apoptosis of certain types of target cells.
This protein thus is designated TNF Related Apoptosis Inducing Ligand (TRAIL).
Among the types of cells that are killed by contact with TRAIL are cancer cells such as
leukemia, lymphoma, and melanoma cells, and cells infected with a virus.

A method for producing TRAIL polypeptides involves culturing host cells
10 transformed with a recombinant expression vector that contains TRAIL-encoding DNA
under conditions appropriate for expression of TRAIL, then recovering the expressed
TRAIL polypeptide from the culture. Antibodies directed against TRAIL polypeptides
are also provided.

15 BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 presents the results of an assay described in example 8. The assay
demonstrated that a soluble human TRAIL polypeptide induced death of Jurkat cells,
which are a leukemia cell line.

Figure 2 presents the results of an assay described in example 1 1 . Contact with
20 a soluble human TRAIL polypeptide induced death of cytomegalovirus-infected human
fibroblasts, whereas non-virally infected fibroblasts were not killed.

DETAILED DESCRIPTION OF THE INVENTION

A novel protein designated TRAIL is provided herein, along with DNA
25 encoding TRAIL and recombinant expression vectors comprising TRAIL DNA. A

method for producing recombinant TRAIL polypeptides involves cultivating host cells
transformed with the recombinant expression vectors under conditions appropriate for
expression of TRAIL, and recovering the expressed TRAIL.

The present invention also provides antibodies that specifically bind TRAIL
30 proteins. In one embodiment, the antibodies are monoclonal antibodies.

The TRAIL protein induces apoptosis of certain types of target cells, such as
transformed cells that include but are not limited to cancer cells and virally-infected
cells. As demonstrated in examples 5, 8, 9, and 10 below, TRAIL induced apoptosis
of human leukemia, lymphoma, and melanoma cell lines. Among the uses of TRAIL is
35 use in killing cancer cells. TRAIL finds further use in treatment of viral infections.
Infection with cytomegalovirus (CMV) rendered human fibroblasts susceptible to

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apoptosis when contacted with TRAIL, whereas uninfected fibroblasts were not killed
through contact with TRAIL (see example 11).

Isolation of a DNA encoding human TRAIL is described in example 1 below.
The nucleotide sequence of the human TRAIL DNA isolated in example 1 is presented
5 in SEQ ID NO: 1 , and the amino acid sequence encoded thereby is presented in SEQ ID
NO: 2. This human TRAIL protein comprises an N-terminal cytoplasmic domain
(amino acids 1-18), a transmembrane region (amino acids 19-38), and an extracellular
domain (amino acids 39-281). The extracellular domain contains a receptor-binding
region.

10 E. coli strain DH10B cells transformed with a recombinant vector containing

this human TRAIL DNA were deposited with the American Type Culture Collection on
June 14, 1995, and assigned accession no. 69849. The deposit was made under the
terms of the Budapest Treaty. The recombinant vector in the deposited strain is the
expression vector pDC409 (described in example 5). The vector was digested with

1 5 Sail and NotI, and human TRAIL DNA that includes the entire coding region shown in
SEQ ID NO: 1 was ligated into the vector.

DNA encoding a second human TRAIL protein was isolated as described in
example 2. The nucleotide sequence of this DNA is presented in SEQ ID NO:3, and
the amino acid sequence encoded thereby is presented in SEQ ID NO:4. The encoded

20 protein comprises an N-terminal cytoplasmic domain (amino acids 1-18), a

transmembrane region (amino acids 19-38), and an extracellular domain (amino acids
39-101).

The DNA of SEQ ID NO:3 lacks a portion of the DNA of SEQ ID NO: 1, and is
thus designated the human TRAIL deletion variant (huTRAILdv) clone. Nucleotides

25 18 through 358 of SEQ ID NO: 1 are identical to nucleotides 8 through 348 of the

huTRAILdv DNA of SEQ ID NO:3. Nucleotides 359 through 506 of SEQ ID NO: 1
are missing from the cloned DNA of SEQ ID NO:3. The deletion causes a shift in the
reading frame, which results in an in-frame stop codon after amino acid 101 of SEQ ID
NO:4. The DNA of SEQ ID NO:3 thus encodes a truncated protein. Amino acids 1

30 through 90 of SEQ ID NO:2 are identical to amino acids 1 through 90 of SEQ ID NO:4.
However, due to the deletion, the C-terminal portion of the huTRAILdv protein (amino
acids 91 through 101 of SEQ ID NO:4) differs from the residues in the corresponding
positions in SEQ ID NO:2. In contrast to the full length huTRAIL protein, the
truncated huTRAILdv protein does not exhibit the ability to induce apoptosis of the T

35 cell leukemia cells of the Jurkat cell line.

DNA encoding a mouse TRAIL protein has also been isolated, as described in
example 3. The nucleotide sequence of this DNA is presented in SEQ ID NO:5 and the

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amino acid sequence encoded thereby is presented in SEQ ID NO: 6. The encoded
protein comprises an N-terminal cytoplasmic domain (amino acids 1-17), a
transmembrane region (amino acids 18-38), and an extracellular domain (amino acids
39-291). This mouse TRAIL is 64% identical to the human TRAIL of SEQ ID NO:2 at
5 the amino acid level. The coding region of the mouse TRAIL nucleotide sequence is
75% identical to the coding region of the human nucleotide sequence of SEQ ID NO: 1 .

One embodiment of the present invention is directed to human TRAIL protein
characterized by the N-terminal amino acid sequence MetAlaMetMetGlu ValGlnGly
GlyProSerLeuGlyGlnThr (amino acids 1-15 of SEQ ID NOS:2 and 4). Mouse TRAIL
10 proteins characterized by the N-terminal amino acid sequence MetProSerSerGlyAla
LeuLysAspLeuSerPheSerGlnHis (amino acids 1-15 of SEQ ID NO:6) are also
provided herein.

The TRAIL of the present invention is distinct from the protein known as Fas
ligand (Suda et al., Cell, 75:1 169, 1993; Takahashi et al., International Immunology

15 6: 1567, 1994). Fas ligand induces apoptosis of certain cell types, via the receptor

known as Fas. As demonstrated in example 5, TRAIL-induced apoptosis of target cells
is not mediated through Fas. The human TRAIL amino acid sequence of SEQ ID NO:2
is about 20% identical to the human Fas ligand amino acid sequence that is presented in
Takahashi et al., supra. The extracellular domain of human TRAIL is about 28.4%

20 identical to the extracellular domain of human Fas ligand.

The amino acid sequences disclosed herein reveal that TRAIL is a member of
the TNF family of ligands (Smith et al. Cell, 73: 1349, 1993; Suda et al., Cell,
75: 1 169, 1993; Smith et al., Cell, 76:959, 1994). The percent identities between the
human TRAIT, extracellular domain amino acid sequence and the amino acid sequence

25 of the extracellular domain of other proteins of this family are as follows: 28.4% with
Fas ligand, 22.4% with lymphotoxin-p, 22.9% with TNF-a, 23.1% with TNF-p,
22.1% with CD30 ligand, and 23.4% with CD40 ligand.

TRAIL was tested for ability to bind receptors of the TNF-R family of
receptors. The binding analysis was conducted using the slide autoradiography

30 procedure of Gearing et al. (EMBO J. 8:3667, 1989). The analysis revealed no

detectable binding of human TRAIL to human CD30, CD40, 4-1BB, OX40, TNF-R
(p80 form), CD27, or LTpR (also known as TNFR-RP). The results in example 5
indicate that human TRAIL does not bind human Fas.

The TRAIL polypeptides of the present invention include polypeptides having

35 amino acid sequences that differ from, but are highly homologous to, those presented
in SEQ ID NOS:2 and 6. Examples include, but are not limited to, homologs derived
from other mammalian species, variants (both naturally occurring variants and those

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generated by recombinant DNA technology), and TRAIL fragments that retain a desired
biological activity. Such polypeptides exhibit a biological activity of the TRAIL
proteins of SEQ ID NOS:2 and 6, and preferably comprise an amino acid sequence that
is at least 80% identical (most preferably at least 90% identical) to the amino acid
5 sequence presented in SEQ ID NO:2 or SEQ ID NO:6. These embodiments of the
present invention are described in more detail below.

Conserved sequences located in the C-terminal portion of proteins in the TNF
family are identified in Smith et al. (Cell, 73:1349, 1993, see page 1353 and Figure 6);
Suda et al. (Cell, 75:1 169, 1993, see figure 7); Smith et al. (Cell, 76:959, 1994, see
10 figure 3); and Goodwin et al. (Eur. J. Immunol, 23:2631, 1993, see figure 7 and
pages 2638-39), hereby incorporated by reference. Among the amino acids in the
human TRAIL protein that are conserved (in at least a majority of TNF family
members) are those in positions 124-125 (AH), 136 (L), 154 (W), 169 (L), 174 (L),
180 (G), 182 (Y), 187 (Q), 190 (F), 193 (Q), and 275-276 (FG) of SEQ ID NO:2.
1 5 Another structural feature of TRAIL is a spacer region between the C-terminus of the
trans-membrane region and the portion of the extracellular domain that is believed to be
most important for biological activity. This spacer region, located at the N-terminus of
the extracellular domain, consists of amino acids 39 through 94 of SEQ ID NO:2.
Analogous spacers are found in other family members, e.g., CD40 ligand. Amino
20 acids 138 through 153 correspond to a loop between the 6 sheets of the folded (three
dimensional) human TRAIL protein.

Provided herein are membrane-bound TRAIL proteins (comprising a
cytoplasmic domain, a transmembrane region, and an extracellular domain) as well as
TRAIL fragments that retain a desired biological property of the full length TRAIL
25 protein. In one embodiment, TRAIL fragments are soluble TRAIL polypeptides

comprising all or part of the extracellular domain, but lacking the transmembrane region
that would cause retention of the polypeptide on a cell membrane. Soluble TRAIL
proteins are capable of being secreted from the cells in which they are expressed.
Advantageously, a heterologous signal peptide is fused to the N-terminus such that the
30 soluble TRAIL is secreted upon expression.

Soluble TRAIL may be identified (and distinguished from its non-soluble
membrane-bound counterparts) by separating intact cells which express the desired
protein from the culture medium, e.g., by centrifugation, and assaying the medium
(supernatant) for the presence of the desired protein. The presence of TRAIL in the
35 medium indicates that the protein was secreted from the cells and thus is a soluble form
of the TRAIL protein. Naturally-occurring soluble forms of TRAIL are encompassed
by the present invention.

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The use of soluble forms of TRAIL is advantageous for certain applications.
Purification of the proteins from recombinant host cells is facilitated, since the soluble
proteins are secreted from the cells. Further, soluble proteins are generally more
suitable for intravenous administration.
5 Examples of soluble TRAIL polypeptides are those containing the entire

extracellular domain (e.g., amino acids 39 to 281 of SEQ ID NO:2 or amino acids 39 to
291 of SEQ ID NO:6). Fragments of the extracellular domain that retain a desired
biological activity are also provided. Such fragments advantageously include regions
of TRAIL that are conserved in proteins of the TNF family of ligands, as described
10 above.

Additional examples of soluble TRAIL polypeptides are those lacking not only
the cytoplasmic domain and transmembrane region, but also all or part of the above-
described spacer region. Soluble human TRAIL polypeptides thus include, but are not
limited to, polypeptides comprising amino acids x to 28 1 , wherein x represents any of
15 the amino acids in positions 39 through 95 of SEQ ID NO:2. In the embodiment in
which residue 95 is the N-terminal amino acid, the entire spacer region has been
deleted.

TRAIL fragments, including soluble polypeptides, may be prepared by any of a
number of conventional techniques. A DNA sequence encoding a desired TRAIL

20 fragment may be subcloned into an expression vector for production of the TRAIL

fragment. The TRAIL-encoding DNA sequence advantageously is fused to a sequence
encoding a suitable leader or signal peptide. The desired TRAIL-encoding DNA
fragment may be chemically synthesized using known techniques. DNA fragments
also may be produced by restriction endonuclease digestion of a full length cloned DNA

25 sequence, and isolated by electrophoresis on agarose gels. If necessary,

oligonucleotides that reconstruct the 5' or 3' terminus to a desired point may be ligated
to a DNA fragment generated by restriction enzyme digestion. Such oligonucleotides
may additionally contain a restriction endonuclease cleavage site upstream of the desired
coding sequence, and position an initiation codon (ATG) at the N-terminus of the

30 coding sequence.

The well known polymerase chain reaction (PCR) procedure also may be
employed to isolate and amplify a DNA sequence encoding a desired protein fragment.
Oligonucleotides that define the desired termini of the DNA fragment are employed as
5' and 3' primers. The oligonucleotides may additionally contain recognition sites for

35 restriction endonucleases, to faciliate insertion of the amplified DNA fragment into an
expression vector. PCR techniques are described in Saiki et al., Science 239:487
(1988); Recombinant DNA Methodology, Wu et al., eds., Academic Press, Inc., San

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Diego (1989), pp. 189-196; and PCR Protocols: A Guide to Methods and
Applications, Innis et al., eds., Academic Press, Inc. (1990).

As will be understood by the skilled artisan, the transmembrane region of each
TRAIL protein discussed above is identified in accordance with conventional criteria for
5 identifying that type of hydrophobic domain. The exact boundaries of a transmembrane
region may vary slightly (most likely by no more than five amino acids on either end)
from those presented above. Computer programs useful for identifying such
hydrophobic regions in proteins are available.

The TRAIL DN A of the present invention includes cDN A, chemically
10 synthesized DNA, DNA isolated by PCR, genomic DNA, and combinations thereof.
Genomic TRAIL DNA may be isolated by hybridization to the TRAIL cDNA disclosed
herein using standard techniques. RNA transcribed from the TRAIL DNA is also
encompassed by the present invention.

A search of the NCBI databank identified five expressed sequence tags (ESTs)
15 having regions of identity with TRAIL DNA. These ESTs (NCBI accession numbers
T90422, T82085, T10524, R31020, and Z36726) are all human cDNA fragments.
The NCBI records do not disclose any polypeptide encoded by the ESTs, and do not
indicate what the reading frame, if any, might be. However, even if the knowledge of
the reading frame revealed herein by disclosure of complete TRAIL coding regions is
20 used to express the ESTs, none of the encoded polypeptides would have the apoptosis-
inducing property of the presently-claimed TRAIL polypeptides. In other words, if
each of the five ESTs were inserted into expression vectors downstream from an
initiator methionine codon, in the reading frame elucidated herein, none of the resulting
expressed polypeptides would contain a sufficient portion of the extracellular domain of
25 TRAIL to induce apoptosis of Jurkat cells.

Certain embodiments of the present invention provide isolated DNA comprising
a nucleotide sequence selected from the group consisting of nucleotides 88 to 933 of
SEQ ID NO: 1 (human TRAIL coding region); nucleotides 202 to 933 of SEQ ID NO: 1
(encoding the human TRAIL extracellular domain); nucleotides 47 to 922 of SEQ ID
30 NO:5 (mouse TRAIL coding region); and nucleotides 261 to 922 of SEQ ID NO:5

(encoding the mouse TRAIL extracellular domain). DNAs encoding biologically active
fragments of the proteins of SEQ ID NOS:2 and 6 are also provided. Further
embodiments include sequences comprising nucleotides 370 to 930 of SEQ ID NO: 1
and nucleotides 341 to 919 of SEQ ID NO:5, which encode the particular human and
35 murine soluble TRAIL polypeptides, respectively, described in example 7.

Due to degeneracy of the genetic code, two DNA sequences may differ, yet
encode the same amino acid sequence. The present invention thus provides isolated

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DNA sequences encoding biologically active TRAIL, selected from DNA comprising
the coding region of a native human or murine TRAIL cDNA, or fragments thereof,
and DNA which is degenerate as a result of the genetic code to the native TRAIL DNA
sequence.

5 Also provided herein are purified TRAIL polypeptides, both recombinant and

non-recombinant. Variants and derivatives of native TRAIL proteins that retain a
desired biological activity are also within the scope of the present invention. In one
embodiment, the biological activity of an TRAIL variant is essentially equivalent to the
biological activity of a native TRAIL protein. One desired biological activity of TRAIL

10 is the ability to induce death of Jurkat cells. Assay procedures for detecting apoptosis
of target cells are well known. DNA laddering is among the characteristics of cell death
via apoptosis, and is recognized as one of the observable phenomena that distinguish
apoptotic cell death from necrotic cell death. Examples of assay techniques suitable for
detecting death or apoptosis of target cells include those described in examples 5 and 8

15 to 1 1 . Another property of TRAIL is the ability to bind to Jurkat cells.

TRAIL variants may be obtained by mutations of native TRAIL nucleotide
sequences, for example. A TRAIL variant, as referred to herein, is a polypeptide
substantially homologous to a native TRAIL, but which has an amino acid sequence
different from that of native TRAIL because of one or a plurality of deletions, insertions

20 or substitutions. TRAIL-encoding DNA sequences of the present invention encompass
sequences that comprise one or more additions, deletions, or substitutions of
nucleotides when compared to a native TRAIL DNA sequence, but that encode an
TRAIL protein that is essentially biologically equivalent to a native TRAIL protein.

The variant amino acid or DNA sequence preferably is at least 80% identical to a

25 native TRAIL sequence, most preferably at least 90% identical. The degree of
homology (percent identity) between a native and a mutant sequence may be
determined, for example, by comparing the two sequences using computer programs
commonly employed for this purpose. One suitable program is the GAP computer
program, version 6.0, described by Devereux et al. (Nucl. Acids Res. 12:387, 1984)

30 and available from the University of Wisconsin Genetics Computer Group (UWGCG).
The GAP program utilizes the alignment method of Needleman and Wunsch (J. Mol
Biol. 48:443, 1970), as revised by Smith and Waterman {Adv. Appl Math 2:482,
1981). Briefly, the GAP program defines identity as the number of aligned symbols
(i.e., nucleotides or amino acids) which are identical, divided by the total number of

35 symbols in the shorter of the two sequences. The preferred default parameters for the
GAP program include: (1) a unary comparison matrix (containing a value of 1 for
identities and 0 for non-identities) for nucleotides, and the weighted comparison matrix

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of Gribskov and Burgess, NucL Acids Res. 14:6745, 1986, as described by Schwartz
and Dayhoff, eds., Atlas of Protein Sequence and Structure, National Biomedical
Research Foundation, pp. 353-358, 1979; (2) a penalty of 3.0 for each gap and an
additional 0. 10 penalty for each symbol in each gap; and (3) no penalty for end gaps.
5 Alterations of the native amino acid sequence may be accomplished by any of a

number of known techniques. Mutations can be introduced at particular loci by
synthesizing oligonucleotides containing a mutant sequence, flanked by restriction sites
enabling ligation to fragments of the native sequence. Following ligation, the resulting
reconstructed sequence encodes an analog having the desired amino acid insertion,

10 substitution, or deletion.

Alternatively, oligonucleotide-directed site-specific mutagenesis procedures can
be employed to provide an altered gene having particular codons altered according to
the substitution, deletion, or insertion required. Techniques for making such alterations
include those disclosed by Walder et al. {Gene 42: 133, 1986); Bauer et al. {Gene

15 37:73, 1985); Craik {BioTechniques, January 1985, 12-19); Smith et al. {Genetic
Engineering: Principles and Methods, Plenum Press, 1981); and U.S. Patent Nos.
4,518,584 and 4,737,462, which are incorporated by reference herein.

Variants may comprise conservatively substituted sequences, meaning that one
or more amino acid residues of a native TRAIL polypeptide are replaced by different

20 residues, but that the conservatively substituted TRAIL polypeptide retains a desired
biological activity that is essentially equivalent to that of a native TRAIL polypeptide.
Examples of conservative substitutions include substitution of amino acids that do not
alter the secondary and/or tertiary structure of TRAIL. Other examples involve
substitution of amino acids outside of the receptor-binding domain, when the desired

25 biological activity is the ability to bind to a receptor on target cells and induce apoptosis
of the target cells. A given amino acid may be replaced by a residue having similar
physiochemical characteristics, e.g., substituting one aliphatic residue for another (such
as He, Val, Leu, or Ala for one another), or substitution of one polar residue for
another (such as between Lys and Arg; Glu and Asp; or Gin and Asn). Other such

30 conservative substitutions, e.g., substitutions of entire regions having similar

hydrophobicity characteristics, are well known. TRAIL polypeptides comprising
conservative amino acid substitutions may be tested in one of the assays described
herein to confirm that a desired biological activity of a native TRAIL is retained. DN A
sequences encoding TRAIL polypeptides that contain such conservative amino acid

35 substitutions are encompassed by the present invention.

Conserved amino acids located in the C-terminal portion of proteins in the TNF
family, and believed to be important for biological activity, have been identified. These

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conserved sequences are discussed in Smith et al. {Cell, 73:1349, 1993, see page 1353
and Figure 6); Suda et al. {Cell, 75: 1 169, 1993, see figure 7); Smith et al. {Cell
76:959, 1994, see figure 3); and Goodwin et al. {Eur. J. Immunol, 23:2631, 1993,
see figure 7 and pages 2638-39). Advantageously, the conserved amino acids are not
5 altered when generating conservatively substituted sequences. If altered, amino acids
found at equivalent positions in other members of the TNF family are substituted.

TRAIL also may be modified to create TRAIL derivatives by forming covalent
or aggregative conjugates with other chemical moieties, such as glycosyl groups,
lipids, phosphate, acetyl groups and the like. Covalent derivatives of TRAIL may be

10 prepared by linking the chemical moieties to functional groups on TRAIL amino acid
side chains or at the N-terminus or C-terminus of a TRAIL polypeptide or the
extracellular domain thereof. Other derivatives of TRAIL within the scope of this
invention include covalent or aggregative conjugates of TRAIL or its fragments with
other proteins or polypeptides, such as by synthesis in recombinant culture as N-

15 terminal or C-terminal fusions. For example, the conjugate may comprise a signal or
leader polypeptide sequence (e.g. the a-factor leader of Saccharomyces) at the N-
terminus of a TRAIL polypeptide. The signal or leader peptide co-translationally or
post-translationally directs transfer of the conjugate from its site of synthesis to a site
inside or outside of the cell membrane or cell wall.

20 TRAIL polypeptide fusions can comprise peptides added to facilitate

purification and identification of TRAIL. Such peptides include, for example, poly -His
or the antigenic identification peptides described in U.S. Patent No. 5,01 1,912 and in
Hopp et al., Bio/Technology 6:1204, 1988. One such peptide is the FLAG® peptide,
Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys (DYKDDDDK) (SEQ ID NO:7), which is highly

25 antigenic and provides an epitope reversibly bound by a specific monoclonal antibody,
thus enabling rapid assay and facile purification of expressed recombinant protein.
This sequence is also specifically cleaved by bovine mucosal enterokinase at the residue
immediately following the Asp-Lys pairing. Fusion proteins capped with this peptide
may also be resistant to intracellular degradation in E. colL

30 A murine hybridoma designated 4E1 1 produces a monoclonal antibody that

binds the peptide DYKDDDDK (SEQ ID NO:7) in the presence of certain divalent metal
cations (as described in U.S. Patent 5,01 1,912), and has been deposited with the
American Type Culture Collection under accession no HB 9259. Expression systems
useful for producing recombinant proteins fused to the FLAG® peptide, as well as

35 monoclonal antibodies that bind the peptide and are useful in purifying the recombinant
proteins, are available from Eastman Kodak Company, Scientific Imaging Systems,
New Haven, Connecticut.

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The present invention further includes TRAIL polypeptides with or without
associated native-pattern glycosylation. TRAIL expressed in yeast or mammalian
expression systems may be similar to or significantly different from a native TRAIL
polypeptide in molecular weight and glycosylation pattern, depending upon the choice

5 of expression system. Expression of TRAIL polypeptides in bacterial expression
systems, such as E. colU provides non-glycosylated molecules.

Glycosylation sites in the TRAIL extracellular domain can be modified to
preclude glycosylation while allowing expression of a homogeneous, reduced
carbohydrate analog using yeast or mammalian expression systems. N-glycosylation

10 sites in eukaryotic polypeptides are characterized by an amino acid triplet Asn-X-Y,
wherein X is any amino acid except Pro and Y is Ser or Thr. Appropriate
modifications to the nucleotide sequence encoding this triplet will result in
substitutions, additions or deletions that prevent attachment of carbohydrate residues at
the Asn side chain. Known procedures for inactivating N-glycosylation sites in

15 proteins include those described in U.S. Patent 5,071,972 and EP 276,846. A

potential N-glycosylation site is found at positions 109-1 1 1 in the human protein of
SEQ ID NO:2 and at positions 52-54 in the murine protein of SEQ ID NO:6.

In another example, sequences encoding Cys residues that are not essential for
biological activity can be altered to cause the Cys residues to be deleted or replaced with

20 other amino acids, preventing formation of incorrect intramolecular disulfide bridges
upon renaturation. Other variants are prepared by modification of adjacent dibasic
amino acid residues to enhance expression in yeast systems in which KEX2 protease
activity is present. EP 212,914 discloses the use of site-specific mutagenesis to
inactivate KEX2 protease processing sites in a protein. KEX2 protease processing

25 sites are inactivated by deleting, adding or substituting residues to alter Arg-Arg,
Arg-Lys, and Lys-Arg pairs to eliminate the occurrence of these adjacent basic
residues. Lys-Lys pairings are considerably less susceptible to KEX2 cleavage, and
conversion of Arg-Lys or Lys-Arg to Lys-Lys represents a conservative and preferred
approach to inactivating KEX2 sites. Potential KEX2 protease processing sites are

30 found at positions 89-90 and 149-150 in the protein of SEQ ID NO:2, and at positions
85-86, 135-136, and 162-163 in the protein of SEQ ID NO:6.

Naturally occurring TRAIL variants are also encompassed by the present
invention. Examples of such variants are proteins that result from alternative mRNA
splicing events (since TRAIL is encoded by a multi-exon gene) or from proteolytic

35 cleavage of the TRAIL protein, wherein a desired biological activity is retained.

Alternative splicing of mRNA may yield a truncated but biologically active TRAIL
protein, such as a naturally occurring soluble form of the protein, for example.

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Variations attributable to proteolysis include, for example, differences in the N- or O
termini upon expression in different types of host cells, due to proteolytic removal of
one or more terminal amino acids from the TRAIL protein. In addition, proteolytic
cleavage may release a soluble form of TRAIL from a membrane-bound form of the
5 protein. Allelic variants are also encompassed by the present invention.

Oligomers

The present invention encompasses TRAIL polypeptides in the form of
oligomers, such as dimers, trimers, or higher oligomers. Oligomers may be formed by

10 disulfide bonds between cysteine residues on different TRAIL polypeptides, or by non-
covalent interactions between TRAIL polypeptide chains, for example. In other
embodiments, oligomers comprise from two to four TRAIL polypeptides joined via
covalent or non-covalent interactions between peptide moieties fused to the TRAIL
polypeptides. Such peptides may be peptide linkers (spacers), or peptides that have the

15 property of promoting oligomerization. Leucine zippers and certain polypeptides
derived from antibodies are among the peptides that can promote oligomerization of
TRAIL polypeptides attached thereto, as described in more detail below. The TRAIL
polypeptides preferably are soluble.

Preparation of fusion proteins comprising heterologous polypeptides fused to

20 various portions of antibody-derived polypeptides (including the Fc domain) has been
described, e.g., by Ashkenazi et al. (PNAS USA 88:10535, 1991); Byrn et al. (Nature
344:667, 1990); and Hollenbaugh and Aruffo ("Construction of Immunoglobulin
Fusion Proteins", in Current Protocols in Immunology, Supplement 4, pages 10.19.1 -
10.19.1 1, 1992), hereby incorporated by reference. In one embodiment of the

25 invention, an TRAIL dimer is created by fusing TRAIL to an Fc region polypeptide
derived from an antibody. The term "Fc polypeptide" includes native and mutein
forms, as well as truncated Fc polypeptides containing the hinge region that promotes
dimerization. The Fc polypeptide preferably is fused to a soluble TRAIL (e.g.,
comprising only the extracellular domain).

30 A gene fusion encoding the TRAIL/Fc fusion protein is inserted into an

appropriate expression vector. The TRAIL/Fc fusion proteins are allowed to assemble
much like antibody molecules, whereupon interchain disulfide bonds form between the

Fc polypeptides, yielding divalent TRAIL. In other embodiments, TRAIL may be
substituted for the variable portion of an antibody heavy or light chain. If fusion
35 proteins are made with both heavy and light chains of an antibody, it is possible to form
an TRAIL oligomer with as many as four TRAIL extracellular regions.

WO 97/01633 PCT/US96/10895

One suitable Fc polypeptide is the native Fc region polypeptide derived from a
human IgGl, which is described in PCT application WO 93/10151, hereby
incorporated by reference. Another useful Fc polypeptide is the Fc mutein described in
U.S. Patent 5,457,035. The amino acid sequence of the mutein is identical to that of
5 the native Fc sequence presented in WO 93/10151, except that amino acid 19 has been
changed from Leu to Ala, amino acid 20 has been changed from Leu to Glu, and amino
acid 22 has been changed from Gly to Ala. This mutein Fc exhibits reduced affinity for

immunoglobulin receptors.

Alternatively, oligomeric TRAIL may comprise two or more soluble TRAIL

10 polypeptides joined through peptide linkers. Examples include those peptide linkers
described in United States Patent 5,073,627 (hereby incorporated by reference).
Fusion proteins comprising multiple TRAIL polypeptides separated by peptide linkers
may be produced using conventional recombinant DNA technology.

Another method for preparing oligomeric TRAIL polypeptides involves use of a

1 5 leucine zipper. Leucine zipper domains are peptides that promote oligomerization of the
proteins in which they are found. Leucine zippers were originally identified in several
DNA-binding proteins (Landschulz et al., Science 240:1759, 1988), and have since
been found in a variety of different proteins. Among the known leucine zippers are
naturally occurring peptides and derivatives thereof that dimerize or trimerize.

20 Examples of leucine zipper domains suitable for producing soluble oligomeric TRAIL
proteins are those described PCT application WO 94/10308, hereby incorporated by
reference. Recombinant fusion proteins comprising a soluble TRAIL polypeptide fused
to a peptide that dimerizes or trimerizes in solution are expressed in suitable host cells,
and the resulting soluble oligomeric TRAIL is recovered from the culture supernatant.

25 Certain members of the TNF family of proteins are believed to exist in trimeric

form (Beutler and Huffel, Science 264:667, 1994; Banner et al., Cell 73:431, 1993).
Thus, trimeric TRAIL may offer the advantage of enhanced biological activity.
Preferred leucine zipper moieties are those that preferentially form trimers. One
example is a leucine zipper derived from lung surfactant protein D (SPD), as described

30 in Hoppe et al. (FEBS Letters 344: 191, 1994) and in U.S. Patent application serial no.
08/446,922, hereby incorporated by reference. Other peptides derived from naturally
occurring trimeric proteins may be employed in preparing trimeric TRAIL.

As described in example 7, a soluble Flag®-TRAIL polypeptide expressed in
CV-1/EBNA cells spontaneously formed oligomers believed to be a mixture of dimers

35 and trimers. The cytotoxic effect of this soluble Flag®-TRAIL in the assay of example
8 was enhanced by including an anti-Flag® antibody, possibly because the antibody
facilitated cross-linking of TRAIL/receptor complexes. In one embodiment of the

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invention, biological activity of TRAIL is enhanced by employing TRAIL in
conjunction with an antibody that is capable of cross-linking TRAIL. Cells that are to
be killed may be contacted with both a soluble TRAIL polypeptide and such an
antibody.

5 As one example, cancer or virally infected cells are contacted with an anti-Flag®

antibody and a soluble Flag®~TRAIL polypeptide. Preferably, an antibody fragment
lacking the Fc region is employed. Bivalent forms of the antibody may bind the Flag®
moieties of two soluble Flag®-TRAIL polypeptides that are found in separate dimers or
trimers. The antibody may be mixed or incubated with a Flag®-TRAIL polypeptide
10 prior to administration in vivo.

Expression Systems

The present invention provides recombinant expression vectors for expression
of TRAIL, and host cells transformed with the expression vectors. Any suitable

1 5 expression system may be employed. The vectors include a DNA encoding a TRAIL
polypeptide, operably linked to suitable transcriptional or translational regulatory
nucleotide sequences, such as those derived from a mammalian, microbial, viral, or
insect gene. Examples of regulatory sequences include transcriptional promoters,
operators, or enhancers, an mRNA ribosomal binding site, and appropriate sequences

20 which control transcription and translation initiation and termination. Nucleotide

sequences are operably linked when the regulatory sequence functionally relates to the
TRAIL DNA sequence. Thus, a promoter nucleotide sequence is operably linked to an
TRAIL DNA sequence if the promoter nucleotide sequence controls the transcription of
the TRAIL DNA sequence. An origin of replication that confers the ability to replicate

25 in the desired host cells, and a selection gene by which transformants are identified, are
generally incorporated into the expression vector.

In addition, a sequence encoding an appropriate signal peptide can be
incorporated into expression vectors. A DNA sequence for a signal peptide (secretory
leader) may be fused in frame to the TRAIL sequence so that the TRAIL is initially

30 translated as a fusion protein comprising the signal peptide. A signal peptide that is
functional in the intended host cells promotes extracellular secretion of the TRAIL
polypeptide. The signal peptide is cleaved from the TRAIL polypeptide upon secretion
of TRAIL from the cell.

Suitable host cells for expression of TRAIL polypeptides include prokaryotes,

35 yeast or higher eukaryotic cells. Appropriate cloning and expression vectors for use
with bacterial, fungal, yeast, and mammalian cellular hosts are described, for example,
in Pouwels et al. Cloning Vectors: A Laboratory Manual Elsevier, New York,

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(1985). Cell-free translation systems could also be employed to produce TRAIL
polypeptides using RNAs derived from DNA constructs disclosed herein.

Prokaryotes include gram negative or gram positive organisms, for example, E.
coli or Bacilli Suitable prokaryotic host cells for transformation include, for example,

5 E. coli, Bacillus subtilis, Salmonella typhimurium, and various other species within the
genera Pseudomonas, Streptomyces, and Staphylococcus, In a prokaryotic host cell,
such as E. coli, a TRAIL polypeptide may include an N-terminal methionine residue to
facilitate expression of the recombinant polypeptide in the prokaryotic host cell. The N-
terminal Met may be cleaved from the expressed recombinant TRAIL polypeptide.

10 Expression vectors for use in prokaryotic host cells generally comprise one or

more phenotypic selectable marker genes. A phenotypic selectable marker gene is, for
example, a gene encoding a protein that confers antibiotic resistance or that supplies an
autotrophic requirement. Examples of useful expression vectors for prokaryotic host
cells include those derived from commercially available plasmids such as the cloning

15 vector pBR322 (ATCC 37017). pBR322 contains genes for ampicillin and tetracycline
resistance and thus provides simple means for identifying transformed cells. An
appropriate promoter and a TRAIL DNA sequence are inserted into the pBR322 vector.
Other commercially available vectors include, for example, pKK223-3 (Pharmacia Fine
Chemicals, Uppsala, Sweden) and pGEMl (Promega Biotec, Madison, WI, USA).

20 Promoter sequences commonly used for recombinant prokaryotic host cell

expression vectors include p-lactamase (penicillinase), lactose promoter system (Chang
et aL, Nature 275:615, 1978; and Goeddel et aL, Nature 281:544, 1979), tryptophan
(trp) promoter system (Goeddel et aL, NucL Acids Res. 8:4057, 1980; and EP-A-
36776) and tac promoter (Maniatis, Molecular Cloning: A Laboratory Manual, Cold

25 Spring Harbor Laboratory, p. 412, 1982). A particularly useful prokaryotic host cell
expression system employs a phage X Pl promoter and a cI857ts thermolabile repressor
sequence. Plasmid vectors available from the American Type Culture Collection which
incorporate derivatives of the X Pl promoter include plasmid pHUB2 (resident in E.
coli strain JMB9, ATCC 37092) and pPLc28 (resident in E. coli RR1, ATCC 53082).

30 TRAIL alternatively may be expressed in yeast host cells, preferably from the

Saccharomyces genus (e.g., 5. cerevisiae). Other genera of yeast, such as Pichia or
Kluyveromyces, may also be employed. Yeast vectors will often contain an origin of
replication sequence from a 2\i yeast plasmid, an autonomously replicating sequence
(ARS), a promoter region, sequences for polyadenylation, sequences for transcription
35 termination, and a selectable marker gene. Suitable promoter sequences for yeast
vectors include, among others, promoters for metallothionein, 3-phosphoglycerate
kinase (Hitzeman et aL, J. Biol Chem. 255:2073, 1980) or other glycolytic enzymes

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(Hess et al., J. Adv. Enzyme Reg. 7:149, 1968; and Holland et al., Biochem. 77:4900,
1978), such as enolase, glyceraldehyde-3 -phosphate dehydrogenase, hexokinase,
pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-
phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phospho-
5 glucose isomerase, and glucokinase. Other suitable vectors and promoters for use in
yeast expression are further described in Hitzeman, EPA-73,657. Another alternative
is the glucose-repressible ADH2 promoter described by Russell et al. (J. Biol. Chem.
258:261 A, 1982) and Beier et al. {Nature 300:724, 1982). Shuttle vectors replicable in
both yeast and E. coli may be constructed by inserting DNA sequences from pBR322

10 for selection and replication in E. coli (Amp 1 * gene and origin of replication) into the
above-described yeast vectors.

The yeast a-factor leader sequence may be employed to direct secretion of the
TRAIL polypeptide. The a-factor leader sequence is often inserted between the
promoter sequence and the structural gene sequence. See, e.g., Kurjan et al., Cell

15 30:933, 1982 and Bitter et al., Proc. Natl Acad. Set USA 57:5330, 1984. Other
leader sequences suitable for facilitating secretion of recombinant polypeptides from
yeast hosts are known to those of skill in the art. A leader sequence may be modified
near its 3' end to contain one or more restriction sites. This will facilitate fusion of the
leader sequence to the structural gene.

20 Yeast transformation protocols are known to those of skill in the art. One such

protocol is described by Hinnen et al., Proc. Natl Acad. Scl USA 75:1929, 1978.
The Hinnen et al. protocol selects for Trp+ transformants in a selective medium,
wherein the selective medium consists of 0.67% yeast nitrogen base, 0.5% casamino
acids, 2% glucose, 10 (Ig/ml adenine and 20 [ig/nd. uracil.

25 Yeast host cells transformed by vectors containing an ADH2 promoter sequence

may be grown for inducing expression in a "rich" medium. An example of a rich
medium is one consisting of 1% yeast extract, 2% peptone, and 1% glucose
supplemented with 80 fig/ml adenine and 80 (ig/ml uracil. Derepression of the ADH2
promoter occurs when glucose is exhausted from the medium.

30 Mammalian or insect host cell culture systems could also be employed to

express recombinant TRAIL polypeptides. Bacculovirus systems for production of
heterologous proteins in insect cells are reviewed by Luckow and Summers,
Bio/Technology 6:47 (1988). Established cell lines of mammalian origin also may be
employed. Examples of suitable mammalian host cell lines include the COS -7 line of

35 monkey kidney cells (ATCC CRL 1651) (Gluzman et al., Cell 23:175, 1981), L cells,
C127 cells, 3T3 cells (ATCC CCL 163), Chinese hamster ovary (CHO) cells, HeLa
cells, and BHK (ATCC CRL 10) cell lines, and the CVI/EBNA cell line derived from

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the African green monkey kidney cell line CVI (ATCC CCL 70) as described by
McMahan et al. (EMBO J. 10: 2821, 1991).

Transcriptional and translational control sequences for mammalian host cell
expression vectors may be excised from viral genomes. Commonly used promoter
5 sequences and enhancer sequences are derived from Polyoma virus, Adenovirus 2,
Simian Virus 40 (SV40), and human cytomegalovirus. DNA sequences derived from
the S V40 viral genome, for example, SV40 origin, early and late promoter, enhancer,
splice, and polyadenylation sites may be used to provide other genetic elements for
expression of a structural gene sequence in a mammalian host cell. Viral early and late

10 promoters are particularly useful because both are easily obtained from a viral genome
as a fragment which may also contain a viral origin of replication (Fiers et al., Nature
273: 1 13, 1978). Smaller or larger SV40 fragments may also be used, provided the
approximately 250 bp sequence extending from the Hind III site toward the Bgl I site
located in the S V40 viral origin of replication site is included.

1 5 Expression vectors for use in mammalian host cells can be constructed as

disclosed by Okayama and Berg (Mol. Cell. Biol. 5:280, 1983), for example. A useful
system for stable high level expression of mammalian cDNAs in CI 27 murine
mammary epithelial cells can be constructed substantially as described by Cosman et al.
(Mol. Immunol. 23:935, 1986). A high expression vector, PMLSV N1/N4, described

20 by Cosman et al., Nature 372:768, 1984 has been deposited as ATCC 39890.

Additional mammalian expression vectors are described in EP-A-0367566, and in WO
91/18982. As one alternative, the vector may be derived from a retrovirus. Additional
suitable expression systems are described in the examples below.

One preferred expression system employs Chinese hamster ovary (CHO) cells

25 and an expression vector designated PG5.7. This expression vector is described in

U.S. patent application serial no. 08/586,509, filed January 11, 1996, which is hereby
incorporated by reference. PG5.7 components include a fragment of CHO cell
genomic DNA, followed by a CMV-derived promoter, which is followed by a
sequence encoding an adenovirus tripartite leader, which in turn is followed by a

30 sequence encoding dihydrofolate reductase (DHFR). These components were inserted
into the plasmid vector pGEMl (Promega, Madison, WI). DNA encoding a TRAIL
polypeptide (or fuion protein containing TRAIL) may be inserted between the
sequences encoding the tripartite leader and DHFR. Methotrexate may be added to the
culture medium to increase expression levels, as is recognized in the field.

35 The fragment of CHO cell genomic DNA in vector PG5.7 enhances expression

of TRAIL. A phage lysate containing a fragment of genomic DNA isolated from CHO
cells was deposited with the American Type Culture Collection on January 4, 1996,

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and assigned accession number ATCC 9741 1 . Vector PG5.7 contains nucleotides
8671 through 14507 of the CHO genomic DNA insert in strain deposit ATCC 9741 1.

For expression of TRAIL, a type II protein lacking a native signal sequence, a
heterologous signal sequence or leader functional in mammalian host cells may be
5 added. Examples include the signal sequence for interleukin-7 (IL-7) described in
United States Patent 4,965,195, the signal sequence for interleukin-2 receptor
described in Cosman et aL, Nature 312:768 (1984); the interleukin-4 receptor signal
peptide described in EP 367,566; the type I interleukin- 1 receptor signal peptide
described in U.S. Patent 4,968,607; and the type II interleukin- 1 receptor signal

10 peptide described in EP 460,846.

A preferred expression system employs a leader sequence derived from
cytomegalovirus (CMV). Example 7 illustrates the use of one such leader. In example
7, mammalian host cells were transformed with an expression vector encoding the
peptide Met Ala Arg Arg Leu Trp He Leu Ser Leu Leu Ala Val Thr Leu Thr Val Ala Leu

15 Ala Ala Pro Ser Gin Lys Ser Lys Arg Arg Thr Ser Ser (SEQ ID NO:9) fused to the N-
terminus of an octapeptide designated FLAG® (SEQ ID NO:7, described above),
which in turn is fused to the N-terminus of a soluble TRAIL polypeptide. Residues 1
through 29 of SEQ ID NO:9 constitute a CMV-derived leader sequence, whereas
residues 30 through 32 are encoded by oligonucleotides employed in constructing the

20 expression vector described in example 7. In one embodiment, DNA encoding a poly-
His peptide (e.g., a peptide containing six histidine residues) is positioned between the
sequences encoding the CMV leader and the FLAG® peptide.

Expression systems that employ such CMV-derived leader peptides are useful
for expressing proteins other than TRAIL. Expression vectors comprising a DNA

25 sequence that encodes amino acids 1 through 29 of SEQ ID NO:9 are provided herein.
In another embodiment, the vector comprises a sequence that encodes amino acids 1
through 28 of SEQ ID NO:9. DNA encoding a desired heterologous protein is
positioned downstream of, and in the same reading frame as, DNA encoding the leader.
Additional residues (e.g., those encoded by linkers or primers) may be encoded by

30 DNA positioned between the sequences encoding the leader and the desired

heterologous protein, as illustrated by the vector described in example 7. As is
understood in the pertinent field, the expression vectors comprise promoters and any
other desired regulatory sequences, operably linked to the sequences encoding the
leader and heterologous protein.

35 The leader peptide presented in SEQ ID NO:9 may be cleaved after the arginine

residue at position 29 to yield the mature secreted form of a protein fused thereto.

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Alternatively or additionally, cleavage may occur between amino acids 20 and 21, or
between amino acids 28 and 29, of SEQ ID NO:9.

The skilled artisan will recognize that the position(s) at which the signal peptide
is cleaved may vary according to such factors as the type of host cells employed,

5 whether murine or human TRAIL is expressed by the vector, and the like. Analysis by
computer program reveals that the primary cleavage site may be between residues 20
and 21 of SEQ ID NO:9. Cleavage between residues 22 and 23, and between residues
27 and 28, is predicted to be possible, as well. To illustrate, expression and secretion
of a soluble murine TRAIL polypeptide resulted in cleavage of a CMV-derived signal

10 peptide at multiple positions. The three most prominent species of secreted protein (in
descending order) resulted from cleavage between amino acids 20 and 21 of SEQ ID
NO:9, cleavage between amino acids 22 and 23, and cleavage between amino acids 27
and 28.

A method for producing a heterologous recombinant protein involves culturing
1 5 mammalian host cells transformed with such an expression vector under conditions that
promote expression and secretion of the heterologous protein, and recovering the
protein from the culture medium. Expression systems employing CMV leaders may be
used to produce any desired protein, examples of which include, but are not limited to,
colony stimulating factors, interferons, interleukins, other cytokines, and cytokine
20 receptors.

Purified TRAIL Protein

The present invention provides purified TRAIL proteins, which may be
produced by recombinant expression systems as described above or purified from

25 naturally occurring cells. The desired degree of purity may depend on the intended use
of the protein. A relatively high degree of purity is desired when the protein is to be
administered in vivo, for example. Advantageously, TRAIL polypeptides are purified
such that no protein bands corresponding to other proteins are detectable by SDS-
polyacrylamide gel electrophoresis (SDS-PAGE). It will be recognized by one skilled

30 in the pertinent field that multiple bands corresponding to TRAIL protein may be
detected by SDS-PAGE, due to differential glycosylation, variations in post-
translational processing, and the like, as discussed above. A preparation of TRAIL
protein is considered to be purified as long as no bands corresponding to different
(non-TRAIL) proteins are visualized. TRAIL most preferably is purified to substantial

35 homogeneity, as indicated by a single protein band upon analysis by SDS-PAGE. The
protein band may be visualized by silver staining, Coomassie blue staining, or (if the
protein is radiolabeled) by autoradiography.

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One process for producing the TRAIL protein comprises culturing a host cell
transformed with an expression vector comprising a DN A sequence that encodes
TRAIL under conditions such that TRAIL is expressed. The TRAIL protein is then
recovered from the culture (from the culture medium or cell extracts). As the skilled
5 artisan will recognize, procedures for purifying the recombinant TRAIL will vary
according to such factors as the type of host cells employed and whether or not the
TRAIL is secreted into the culture medium.

For example, when expression systems that secrete the recombinant protein are
employed, the culture medium first may be concentrated using a commercially available

10 protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration
unit. Following the concentration step, the concentrate can be applied to a purification
matrix such as a gel filtration medium. Alternatively, an anion exchange resin can be
employed, for example, a matrix or substrate having pendant diethylaminoethyl
(DEAE) groups. The matrices can be acrylamide, agarose, dextran, cellulose or other

15 types commonly employed in protein purification. Alternatively, a cation exchange step
can be employed. Suitable cation exchangers include various insoluble matrices
comprising sulfopropyl or carboxymethyl groups. Sulfopropyl groups are preferred.
Finally, one or more reversed-phase high performance liquid chromatography (RP-
HPLC) steps employing hydrophobic RP-HPLC media, (e.g., silica gel having

20 pendant methyl or other aliphatic groups) can be employed to further purify TRAIL.
Some or all of the foregoing purification steps, in various combinations, can be
employed to provide a purified TRAIL protein.

Recombinant protein produced in bacterial culture may be isolated by initial
disruption of the host cells, centrifugation, extraction from cell pellets if an insoluble

25 polypeptide, or from the supernatant fluid if a soluble polypeptide, followed by one or
more concentration, salting-out, ion exchange, affinity purification or size exclusion
chromatography steps. Finally, RP-HPLC can be employed for final purification
steps. Microbial cells can be disrupted by any convenient method, including freeze-
thaw cycling, sonication, mechanical disruption, or use of cell lysing agents.

30 Transformed yeast host cells are preferably employed to express TRAIL as a

secreted polypeptide. This simplifies purification. Secreted recombinant polypeptide
from a yeast host cell fermentation can be purified by methods analogous to those
disclosed by Urdal et al. (J. Chromatog. 296:171, 1984). Urdal et al. describe two
sequential, reversed-phase HPLC steps for purification of recombinant human IL-2 on

35 a preparative HPLC column.

Alternatively, TRAIL polypeptides can be purified by immunoaffinity
chromatography. An affinity column containing an antibody that binds TRAIL may be

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prepared by conventional procedures and employed in purifying TRAIL. Example 4
describes a procedure for generating monoclonal antibodies directed against TRAIL.

Properties and Uses of TRAIL

5 Programmed cell death (apoptosis) occurs during embryogenesis,

metamorphosis, endocrine-dependent tissue atrophy, normal tissue turnover, and death
of immune thymocytes. Regulation of programmed cell death is vital for normal
functioning of the immune system. To illustrate, T cells that recognize self-antigens are
destroyed through the apoptotic process during maturation of T-cells in the thymus,

10 whereas other T cells are positively selected. The possibility that some T-cells

recognizing certain self epitopes (e.g., inefficiently processed and presented antigenic
determinants of a given self protein) escape this elimination process and subsequently
play a role in autoimmune diseases has been proposed (Gammon et al., Immunology
Today 12:193, 1991).

15 Insufficient apoptosis has been implicated in certain conditions, while elevated

levels of apoptotic cell death have been associated with other diseases. The desirability
of identifying and using agents that regulate apoptosis in treating such disorders is
recognized (Kromer, Advances in Immunology, 58:21 1, 1995; Groux et al., J. Exp.
Med. 175:331, 1992; Sachs and Lotem, Blood 82:15, 1993).

20 Abnormal resistance of T cells toward undergoing apoptosis has been linked to

lymphocytosis, lymphadenopathy, splenomegaly, accumulation of self-reactive T cells,
autoimmune disease, development of leukemia, and development of lymphoma
(Kromer, supra; see especially pages 214-215). Conversely, excessive apoptosis of T
cells has been suggested to play a role in lymphopenia, systemic immunodeficiency,

25 and specific immunodeficiency, with specific examples being virus-induced

immunodeficient states associated with infectious mononucleosis and cytomegalovirus
infection, and tumor-mediated immunosuppression (Kromer, supra; see especially page
214). Depletion of CD4+ T cells in HIV-infected individuals may be attributable to
inappropriate activation-induced cell death (AICD) by apoptosis (Groux et al., J. Exp.

30 Med. 175:331, 1992).

As demonstrated in examples 5 and 8, TRAIL induces apoptosis of the acute T

cell leukemia cell line designated Jurkat clone E6-1 . TRAIL thus is a research reagent

useful in studies of apoptosis, including the regulation of programmed cell death.

Since Jurkat cells are a leukemia cell line arising from T cells, the TRAIL of the present
35 invention finds use in studies of the role TRAIL may play in apoptosis of other

transformed T cells, such as other malignant cell types arising from T cells.

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TRAIL binds Jurkat cells, as well as inducing apoptosis thereof. TRAIL did
not cause death of freshly isolated murine thymocytes, or peripheral blood T cells
(PBTs) freshly extracted from a healthy human donor. A number of uses flow from

these properties of TRAIL.
5 TRAIL polypeptides may be used to purify leukemia cells, or any other cell type

to which TRAIL binds. Leukemia cells may be isolated from a patient's blood, for
example. In one embodiment, the cells are purified by affinity chromatography, using
a chromatography matrix having TRAIL bound thereto. The TRAIL attached to the
chromatography matrix may be a full length protein, an TRAIL fragment comprising

10 the extracellular domain, an TRAIL-containing fusion protein, or other suitable TRAIL
polypeptide described herein. In one embodiment, a soluble TRAIL/Fc fusion protein
is bound to a Protein A or Protein G column through interaction of the Fc moiety with
the Protein A or Protein G. Alternatively, TRAIL may be used in isolating leukemia
cells by flow cytometry.

1 5 The thus-purified leukemia cells are expected to die following binding of

TRAIL, but the dead cells will still bear cell surface antigens, and may be employed as
immunogens in deriving anti-leukemia antibodies. The leukemia cells, or a desired cell
surface antigen isolated therefrom, find further use in vaccine development.

Since TRAIL binds and kills leukemia cells (the Jurkat cell line), TRAIL also

20 may be useful in treating leukemia. A therapeutic method involves contacting leukemia
cells with an effective amount of TRAIL. In one embodiment, a leukemia patient's
blood is contacted ex vivo with an TRAIL polypeptide. The TRAIL may be
immobilized on a suitable matrix. TRAIL binds the leukemia cells, thus removing them
from the patient's blood before the blood is returned into the patient.

25 Alternatively or additionally, bone marrow extracted from a leukemia patient

may be contacted with an amount of TRAIL effective in inducing death of leukemia
cells in the bone marrow. Bone marrow may be aspirated from the sternum or iliac
crests, for example, and contacted with TRAIL to purge leukemia cells. The thus-
treated marrow is returned to the patient.

30 TRAIL also binds to, and induces apoptosis of, lymphoma and melanoma cells

(see examples 5, 9, and 10). Thus, uses of TRAIL that are analogous to those
described above for leukemia cells are applicable to lymphoma and melanoma cells.
TRAIL polypeptides may be employed in treating cancer, including, but not limited to,
leukemia, lymphoma, and melanoma. In one embodiment, the lymphoma is Burkitt's

35 lymphoma. Table I in example 9 shows that TRAIL had a cytotoxic effect on several
Burkitt's lymphoma cell lines. Epstein-Barr virus is an etiologic agent of Burkitt's
lymphoma.

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TRAIL polypeptides also find use in treating viral infections. Contact with
TRAIL caused death of cells infected with cytomegalovirus, but not of the same cell
type when uninfected, as described in example 1 1 . The ability of TRAIL to kill cells
infected with other viruses can be confirmed using the assay described in example 1 1.

5 Such viruses include, but are not limited to, encephalomyocarditis virus, Newcastle
disease virus, vesicular stomatitis virus, herpes simplex virus, adenovirus-2, bovine
viral diarrhea virus, HIV, and Epstein-Barr virus.

An effective amount of TRAIL is administered to a mammal, including a
human, afflicted with a viral infection. In one embodiment, TRAIL is employed in

10 conjunction with interferon to treat a viral infection. In the experiment described in

example 11, pretreatment of CMV-infected cells with y-interferon enhanced the level of
killing of the infected cells that was mediated by TRAIL. TRAIL may be administered
in conjunction with other agents that exert a cytotoxic effect on cancer cells or virus-
infected cells.

15 In another embodiment, TRAIL is used to kill virally infected cells in cell

preparations, tissues, or organs that are to be transplanted. To illustrate, bone marrow
may be contacted with TRAIL to kill virus infected cells that may be present therein,
before the bone marrow is transplanted into the recipient.

The TRAIL of the present invention may be used in developing treatments for
20 any disorder mediated (directly or indirectly) by defective or insufficient amounts of
TRAIL. A therapeutically effective amount of purified TRAIL protein is administered
to a patient afflicted with such a disorder. Alternatively, TRAIL DNA sequences may
be employed in developing a gene therapy approach to treating such disorders.
Disclosure herein of native TRAIL nucleotide sequences permits the detection of
25 defective TRAIL genes, and the replacement thereof with normal TRAIL-encoding
genes. Defective genes may be detected in in vitro diagnostic assays, and by
comparision of the native TRAIL nucleotide sequence disclosed herein with that of a
TRAIL gene derived from a person suspected of harboring a defect in this gene.
The present invention provides pharmaceutical compositions comprising
30 purified TRAIL and a physiologically acceptable carrier, diluent, or excipient. Suitable
carriers, diluents, and excipients are nontoxic to recipients at the dosages and
concentrations employed. Such compositions may comprise buffers, antioxidants such
as ascorbic acid, low molecular weight (less than about 10 residues) polypeptides,
proteins, amino acids, carbohydrates including glucose, sucrose or dextrins, chelating
35 agents such as EDTA, glutathione and opier stabilizers and excipients commonly

employed in pharmaceutical compositions. Neutral buffered saline or saline mixed with
conspecific serum albumin are among the appropriate diluents. The composition may

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be formulated as a lyophilizate using appropriate excipient solutions (e.g. sucrose) as
diluents.

For therapeutic use, purified proteins of the present invention are administered
to a patient, preferably a human, for treatment in a manner appropriate to the indication.
5 Thus, for example, the pharmaceutical compositions can be administered locally, by
intravenous injection, continuous infusion, sustained release from implants, or other
suitable technique. Appropriate dosages and the frequency of administration will
depend, of course, on such factors as the nature and severity of the indication being
treated, the desired response, the condition of the patient and so forth.

10 The TRAIL protein employed in the pharmaceutical compositions preferably is

purified such that the TRAIL protein is substantially free of other proteins of natural or
endogenous origin, desirably containing less than about 1 % by mass of protein
contaminants residual of production processes. Such compositions, however, can
contain other proteins added as stabilizers, carriers, excipients or co-therapeutics.

15 The TRAIL-encoding DNAs disclosed herein find use in the production of

TRAIL polypeptides, as discussed above. Fragments of the TRAIL nucleotide
sequences are also useful. In one embodiment, such fragments comprise at least about
17 consecutive nucleotides, more preferably at least 30 consecutive nucleotides, of the
human or murine TRAIL DNA disclosed herein. DNA and RNA complements of said

20 fragments are provided herein, along with both single-stranded and double-stranded
forms of the TRAIL DNA of SEQ ID NOS: 1 , 3 and 5.

Among the uses of such TRAIL nucleic acid fragments are use as a probe or as
primers in a polymerase chain reaction (PCR). As one example, a probe corresponding
to the extracellular domain of TRAIL may be employed. The probes find use in

25 detecting the presence of TRAIL nucleic acids in in vitro assays and in such procedures
as Northern and Southern blots. Cell types expressing TRAIL can be identified as
well. Such procedures are well known, and the skilled artisan can choose a probe of
suitable length, depending on the particular intended application. For PCR, 5' and 3'
primers corresponding to the termini of a desired TRAIL DNA sequence are employed

30 to isolate and amplify that sequence, using conventional techniques.

Other useful fragments of the TRAIL nucleic acids are antisense or sense
oligonucleotides comprising a single-stranded nucleic acid sequence (either RNA or
DNA) capable of binding to target TRAIL mRNA (sense) or TRAIL DNA (antisense)
sequences. Such a fragment generally comprises at least about 14 nucleotides,

35 preferably from about 14 to about 30 nucleotides. The ability to create an antisense or a
sense oligonucleotide, based upon a cDNA sequence for a given protein is described in,

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for example, Stein and Cohen, Cancer Res. 48:2659, 1988 and van der Krol et al.,
BioTechniques 6:958, 1988.

Binding of antisense or sense oligonucleotides to target nucleic acid sequences
results in the formation of duplexes that block translation (RN A) or transcription

5 (DNA) by one of several means, including enhanced degradation of the duplexes,

premature termination of transcription or translation, or by other means. The antisense
oligonucleotides thus may be used to block expression of TRAIL proteins.

Antisense or sense oligonucleotides further comprise oligonucleotides having
modified sugar-phosphodiester backbones (or other sugar linkages, such as those

10 described in W09 1/06629) and wherein such sugar linkages are resistant to

endogenous nucleases. Such oligonucleotides with resistant sugar linkages are stable
in vivo (i.e., capable of resisting enzymatic degradation) but retain sequence specificity
to be able to bind to target nucleotide sequences. Other examples of sense or antisense
oligonucleotides include those oligonucleotides which are covalently linked to organic

15 moieties, such as those described in WO 90/10448, and other moieties that increases
affinity of the oligonucleotide for a target nucleic acid sequence, such as poly-(L-
lysine). Further still, intercalating agents, such as ellipticine, and alkylating agents or
metal complexes may be attached to sense or antisense oligonucleotides to modify
binding specificities of the antisense or sense oliginucleotide for the target nucleotide

20 sequence.

Antisense or sense oligonucleotides may be introduced into a cell containing the
target nucleic acid sequence by any gene transfer method, including, for example,
CaP04-mediated DNA transfection, electroporation, or other gene transfer vectors such
as Epstein-Barr virus. Antisense or sense oligonucleotides are preferably introduced

25 into a cell containing the target nucleic acid sequence by insertion of the antisense or
sense oligonucleotide into a suitable retroviral vector, then contacting the cell with the
retrovirus vector containing the inserted sequence, either in vivo or ex vivo. Suitable
retroviral vectors include, but are not limited to, the murine retrovirus M-MuLV, N2 (a
retrovirus derived from M-MuLV), or or the double copy vectors designated DCT5A,

30 DCT5B and DCT5C (see PCT Application WO 90/1 364 1 ). Alternatively, other
promotor sequences may be used to express the oligonucleotide.

Sense or antisense oligonucleotides may also be introduced into a cell
containing the target nucleotide sequence by formation of a conjugate with a ligand
binding molecule, as described in WO 91/04753. Suitable ligand binding molecules

35 include, but are not limited to, cell surface receptors, growth factors, other cytokines,
or other ligands that bind to cell surface receptors. Preferably, conjugation of the
ligand binding molecule does not substantially interfere with the ability of the ligand

WO 97/01633 PCT/US96/10895

binding molecule to bind to its corresponding molecule or receptor, or block entry of
the sense or antisense oligonucleotide or its conjugated version into the cell.

Alternatively, a sense or an antisense oligonucleotide may be introduced into a
cell containing the target nucleic acid sequence by formation of an oligonucleotide-lipid
5 complex, as described in WO 90/10448. The sense or antisense oligonucleotide-lipid
complex is preferably dissociated within the cell by an endogenous lipase.

Antibodies Immunoreactive with TRAIL

The TRAIL proteins of the present invention, or immunogenic fragments

10 thereof, may be employed in generating antibodies. The present invention thus

provides antibodies that specifically bind TRAIL, i.e., the antibodies bind to TRAIL via
the antigen-binding sites of the antibody (as opposed to non-specific binding).

Polyclonal and monoclonal antibodies may be prepared by conventional
techniques. See, for example, Monoclonal Antibodies, Hybridomas: A New

15 Dimension in Biological Analyses, Kennet et al. (eds.), Plenum Press, New York

(1980); and Antibodies: A Laboratory Manual , Harlow and Land (eds.), Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, NY, (1988). Production of
monoclonal antibodies that are immunoreactive with TRAIL is further illustrated in
example 4 below.

20 Antigen-binding fragments of such antibodies, which may be produced by

conventional techniques, are also encompassed by the present invention. Examples of
such fragments include, but are not limited to, Fab, F(ab'), and F(ab')2 fragments.
Antibody fragments and derivatives produced by genetic engineering techniques are
also provided.

25 The monoclonal antibodies of the present invention include chimeric antibodies,

e.g., humanized versions of murine monoclonal antibodies. Such humanized
antibodies may be prepared by known techniques, and offer the advantage of reduced
immunogenicity when the antibodies are administered to humans. In one embodiment,
a humanized monoclonal antibody comprises the variable region of a murine antibody

30 (or just the antigen binding site thereof) and a constant region derived from a human
antibody. Alternatively, a humanized antibody fragment may comprise the antigen
binding site of a murine monoclonal antibody and a variable region fragment (lacking
the antigen-binding site) derived from a human antibody. Procedures for the
production of chimeric and further engineered monoclonal antibodies include those

35 described in Riechmann et al. {Nature 332:323, 1988), Liu et al. (PNAS 84:3439,
1987), Larrick et al. (Bio/Technology 7:934, 1989), and Winter and Harris (TIPS
14:139, May, 1993).

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Among the uses of the antibodies is use in assays to detect the presence of
TRAIL polypeptides, either in vitro or in vivo. The antibodies find further use in
purifying TRAIL by affinity chromatography.

Those antibodies that additionally can block binding of TRAIL to target cells

5 may be used to inhibit a biological activity of TRAIL. A therapeutic method involves in
vivo administration of such an antibody in an amount effective in inhibiting a TRAIL-
mediated biological activity. Disorders mediated or exacerbated by TRAIL, directly or
indirectly, are thus treated. Monoclonal antibodies are generally preferred for use in
such therapeutic methods.

1 0 Antibodies directed against TRAIL may be useful for treating thrombotic

microangiopathies. One such disorder is thrombotic thrombocytopenic purpura (TTP)
(Kwaan, H.C., Semin. HematoL, 24:71, 1987; Thompson et al., Blood, 80:1890,
1992). Increasing TTP-associated mortality rates have been reported by the U.S.
Centers for Disease Control (Torok et al., Am. J. HematoL 50:84, 1995).

1 5 Plasma from patients afflicted with TTP (including HIV+ and HIV patients)

induces apoptosis of human endothelial cells of dermal microvascular origin, but not
large vessel origin (Laurence et al., Blood, 87:3245, April 15, 1996). Plasma of TTP
patients thus is thought to contain one or more factors that directly or indirectly induce
apoptosis. In the assay described in example 13 below, polyclonal antibodies raised

20 against TRAIL inhibited TTP plasma-induced apoptosis of dermal microvascular

endothelial cells. The data presented in example 13 suggest that TRAIL is present in
the serum of TTP patients, and may play a role in inducing apoptosis of microvascular
endothelial cells.

Another thrombotic microangiopathy is hemolytic-uremic syndrome (HUS)
25 (Moake, J.L., Lancet, 343:393, 1994; Melnyk et al., {Arch. Intern. Med., 155:2077,
1995; Thompson et al., supra). One embodiment of the invention is directed to use of
an anti-TRAIL antibody to treat the condition that is often referred to as "adult HUS"
(even though it can strike children as well). A disorder known as childhood/diarrhea-
associated HUS differs in etiology from adult HUS.
30 Other conditions characterized by clotting of small blood vessels may be treated

using anti-TRAIL antibodies. Such conditions include but are not limited to the
following. Cardiac problems seen in about 5-10% of pediatric AIDS patients are
believed to involve clotting of small blood vessels. Breakdown of the microvasculature
in the heart has been reported in multiple sclerosis patients. As a further example,
35 treatment of systemic lupus erythematosus (SLE) is contemplated.

In one embodiment, a patient's blood or plasma is contacted with an anti-
TRABL antibody ex vivo. The antibody (preferably a monoclonal antibody) may be

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bound to a suitable chromatography matrix by conventional procedures. The patient's
blood or plasma flows through a chromatography column containing the antibody
bound to the matrix, before being returned to the patient. The immobilized antibody
binds TRAIL, thus removing TRAIL protein from the patient's blood.
5 In an alternative embodiment, the antibodies are administered in vivo, in which

case blocking antibodies are desirably employed. Such antibodies may be identified
using any suitable assay procedure, such as by testing antibodies for the ability to
inhibit binding of TRAIL to target cells. Alternatively, blocking antibodies may be
identified in assays for the ability to inhibit a biological effect of the binding of TRAIL

10 to target cells. Example 12 illustrates one suitable method of identifying blocking
antibodies, wherein antibodies are assayed for the ability to inhibit TRAIL-mediated
lysis of Jurkat cells.

The present invention thus provides a method for treating a thrombotic
microangiopathy, involving use of an effective amount of an antibody directed against

15 TRAIL. Antibodies of the present invention may be employed in in vivo or ex vivo
procedures, to inhibit TRAIL-mediated damage to (e.g., apoptosis of) microvascular
endothelial cells.

Anti-TRAIL antibodies may be employed in conjunction with other agents
useful in treating a particular disorder. In an in vitro study reported by Laurence et al.

20 {Blood 87:3245, 1996), some reduction of TTP plasma-mediated apoptosis of

microvascular endothelial cells was achieved by using an anti-Fas blocking antibody,
aurintricarboxylic acid, or normal plasma depleted of cryoprecipitate.

Thus, a patient may be treated with an agent that inhibits Fas-ligand-mediated
apoptosis of endothelial cells, in combination with an agent that inhibits TRAIL-

25 mediated apoptosis of endothelial cells. In one embodiment, an anti-TRAIL blocking
antibody and an anti-FAS blocking antibody are both administered to a patient afflicted
with a disorder characterized by thrombotic microangiopathy, such as TTP or HUS.
Examples of blocking monoclonal antibodies directed against Fas antigen (CD95) are
described in PCT application publication number WO 95/10540, hereby incorporated

30 by reference.

Pharmaceutical compositions comprising an antibody that is immunoreactive

with TRAIL, and a suitable, diluent, excipient, or carrier, are provided herein. Suitable

components of such compositions are as described above for the compositions

containing TRAIL proteins .
35 The following examples are provided to illustrate particular embodiments of the

present invention, and are not to be construed as limiting the scope of the invention.

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FX AMPLE 1: Isolation of a Human TRAIL DNA

DN A encoding a human TRAIL protein of the present invention was isolated by
the following procedure. A TBLASTN search of the dbEST data base at the National

5 Center for Biological Information (NCBI) was performed, using the query sequence
LWXXXGLYYVYXQVXF (SEQ ID NO: 8). This sequence is based upon the most
conserved region of the TNF ligand family (Smith et al., Cell 73:1349, 1993). An
expressed sequence tag (EST) file, GenBank accession number Z36726, was identified
using these search parameters. The GenBank file indicated that this EST was obtained

10 from a human heart atrium cDNA library.

Two 30-bp oligonucleotides based upon sequences from the 3' and 5' ends of
this EST file were synthesized. The oligonucleotide from the 3' end had the sequence
TGAAATCGAAAGTATGTTTGGGAATAGATG (complement of nucleotides 636 to
665 of SEQ ID NO: 1) and the 5' oligonucleotide was TGACGAAGAGAGTATGAA

15 CAGCCCCTGCTG (nucleotides 291 to 320 of SEQ ID NO:l). The oligonucleotides
were 5* end labeled with 32 P y-ATP and polynucleotide kinase. Two XgtlO cDNA
libraries were screened by conventional methods with an equimolar mixture of these
labeled oligonucleotides as probe. One library was a human heart 5' stretch cDNA
library (Stratagene Cloning Systems, La Jolla, CA). The other was a peripheral blood

20 lymphocyte (PBL) library prepared as follows: PBLs were obtained from normal

human volunteers and treated with 10 ng/ml of OKT3 (an anti-CD3 antibody) and 10
ng/ml of human IL-2 for six days. The PBL cells were washed and stimulated with
500 ng/ml of ionomycin (Calbiochem) and 10 ng/ml PMA for 4 hours. Messenger
RNA was isolated from the stimulated PBL cells. cDNA synthesized on the mRNA

25 template was packaged into A.gtl0 phage vectors (Gigapak®, Stratagene Cloning

Systems, La Jolla, CA).

Recombinant phages were plated onto E. coli strain C600-HFL and screened
using standard plaque hybridization techniques. Nitrocellulose filters were lifted from
these plates in duplicate, and hybridized with the 32 P-labeled oligonucleotides
30 overnight at 67°C in a solution of 60 mM Tris pH 8.0, 2 mM EDTA, 5x Denhardt's
Solution, 6x SSC, 1 mg/ml n-lauroyl sarcosine, 0.5% NP40, and 4 jig/ml SS salmon
sperm DNA. The filters were then washed in 3x SSC at 67°C for thirty minutes.

From the heart 5' stretch cDNA library, one positive plaque was obtained out of
approximately one million plaques. This clone did not include the 3' end of the gene.
35 Using the PBL library, approximately 50 positive plaques were obtained out of

500,000 plaques. Fifteen of these first round positive plaques were picked, and the
inserts from the enriched pools were amplified using oligonucleotide primers designed

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to amplify phage inserts. The resulting products were resolved by 1.5% agarose gel
electrophoresis, blotted onto nitrocellulose, and analyzed by standard Southern blot
technique using the 32 P-labeled 30-mer oligonucleotides as probes. The two plaque
picks that produced the largest bands by Southern analysis were purified by secondary
5 screening, and isolated phage plaques were obtained using the same procedures
described above.

DNA from the isolated phages was prepared by the plate lysis method, and the
cDNA inserts were excised with EcoRI, purified by electrophoresis using 1.5%
agarose in Tris-Borate-EDTA buffer, and ligated into the pBluescript® SK(+) plasmid.

10 These inserts were then sequenced by conventional methods, and the resulting
sequences were aligned.

The nucleotide sequence of a human TRAIL DNA is presented in SEQ ID NO: 1
and the amino acid sequence encoded thereby is presented in SEQ ID NO:2. This
human TRAIL protein comprises an N-terminal cytoplasmic domain (amino acids 1 -

15 18), a transmembrane region (amino acids 19-38), and an extracellular domain (amino
acids 39-281). The calculated molecular weight of this protein is 32,508 daltons.

E. coli strain DH10B cells transformed with a recombinant vector containing
this TRAIL DNA were deposited with the American Type Culture Collection on June
14, 1995, and assigned accession no. 69849. The deposit was made under the terms

20 of the Budapest Treaty. The recombinant vector in the deposited strain is the

expression vector pDC409 (described in example 5). The vector was digested with
Sail and NotI, and human TRAIL DNA that includes the entire coding region shown in
SEQ ID NO: 1 was ligated into the digested vector.

25 EXAMPLE 2: Isolation of DNA Encoding a Truncated TRAIL

DNA encoding a second human TRAIL protein was isolated as follows. This
truncated TRAIL does not exhibit the ability to induce apoptosis of Jurkat cells.

PCR analysis, using the 30-mers described in example 1 as the 5' and 3*
primers, indicated that 3 out of 14 of the first round plaque picks in example 1
30 contained shorter forms of the TRAIL DNA. One of the shortened forms of the gene
was isolated, ligated into the pBluescript® SK(+) cloning vector (Stratagene Cloning
Systems, La Jolla, CA) and sequenced.

The nucleotide sequence of this DNA is presented in SEQ ID NO:3. The amino
acid sequence encoded thereby is presented in SEQ ID NO:4. The encoded protein
35 comprises an N-terminal cytoplasmic domain (amino acids 1-18), a transmembrane
region (amino acids 19-38), and an extracellular domain (amino acids 39-101).

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The DNA of SEQ ID NO:3 lacks nucleotides 359 through 506 of the DNA of
SEQ ID NO: 1, and is thus designated the human TRAIL deletion variant (huTRAILdv)
clone. The deletion causes a shift in the reading frame, which results in an in-frame
stop codon after amino acid 101 of SEQ ID NO:4. The DNA of SEQ ID NO:3 thus

5 encodes a truncated protein. Amino acids 1 through 90 of SEQ ID NO: 2 are identical to
amino acids 1 through 90 of SEQ ID NO:4. However, due to the deletion, the C-
terminal portion of the huTRAILdv protein (amino acids 91 through 101 of SEQ ID
NO:4) differs from the residues in the corresponding positions in SEQ ID NO:2.

The huTRAILdv protein lacks the above-described conserved regions found at

10 the C-terminus of members of the TNF family of proteins. The inability of this
huTRAILdv protein to cause apoptotic death of Jurkat cells further confirms the
importance of these conserved regions for biological activity.

FX AMPLE 3: DNA encoding a murine TRAIL

15 DNA encoding a murine TRAIL was isolated by the following procedure. A

cDNA library comprising cDN A derived from the mouse T cell line 7B9 in the vector
XZAP was prepared as described in Mosley et al. (Cell 59:335, 1989). DNA from the
library was transferred onto nitrocellulose filters by conventional techniques.

Human TRAIL DNA probes were used to identify hybridizing mouse cDNAs

20 on the filters. Two separate probes were used, in two rounds of screening. PCR
reaction products about 400bp in length, isolated and amplified using the human
TRAIL DNA as template, were employed as the probe in the first round of screening.
These PCR products consisted of a fragment of the human TRAIL coding region. The
probe used in the second round of screening consisted of the entire coding region of the

25 human TRAIL DNA of SEQ ID NO: 1 . A random primed DNA labeling kit
(Stratagene, La Jolla, CA) was used to radiolabel the probes.

Hybridization was conducted at 37°C in 50% formamide, followed by washing
with 1 x SSC, 0. 1% SDS at 50°C. A mouse cDNA that was positive in both rounds of

screening was isolated.

30 The nucleotide sequence of this DNA is presented in SEQ ID NO: 5 and the

35 at the amino acid level. The coding region of the mouse TRAIL nucleotide sequence is
75% identical to the coding region of the human nucleotide sequence of SEQ ID NO: 1.

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EXAMPLE 4; Antibodies that hind TRAIL

This example illustrates the preparation of monoclonal antibodies that
5 specifically bind TRAIL. Suitable immunogens that may be employed in generating
such antibodies include, but are not limited to, purified TRAIL protein or an
immunogenic fragment thereof (e.g., the extracellular domain), fusion proteins
containing TRAIL polypeptides (e.g., soluble TRAIL/Fc fusion proteins), and cells
expressing recombinant TRAIL on the cell surface.

10 Known techniques for producing monoclonal antibodies include those described

in U.S. Patent 4,41 1,993. Briefly, mice are immunized with TRAIL as an immunogen
emulsified in complete Freund's adjuvant, and injected in amounts ranging from 10-100
jig subcutaneously or intraperitoneally. Ten to twelve days later, the immunized
animals are boosted with additional TRAIL emulsified in incomplete Freund's adjuvant.

15 Mice are periodically boosted thereafter on a weekly to bi-weekly immunization
schedule. Serum samples are periodically taken by retro-orbital bleeding or tail-tip
excision for testing by dot blot assay or ELISA (Enzyme-Linked Immuno-sorbent
Assay) for TRAIL antibodies.

Following detection of an appropriate antibody titer, positive animals are

20 provided one last intravenous injection of TRAIL in saline. Three to four days later, the
animals are sacrificed, spleen cells harvested, and spleen cells are fused to a murine
myeloma cell line such as NS1 or, preferably, P3x63Ag 8.653 (ATCC CRL 1580).
Fusions generate hybridoma cells, which are plated in multiple microliter plates in a
HAT (hypoxanthine, aminopterin and thymidine) selective medium to inhibit

25 proliferation of non-fused cells, myeloma hybrids, and spleen cell hybrids.

The hybridoma cells are screened by ELISA for reactivity against purified
TRAIL by adaptations of the techniques disclosed in Engvall et al. (Immunochem.
8:871, 1971) and in U.S. Patent 4,703,004. Positive hybridoma cells can be injected
intraperitoneally into syngeneic BALB/c mice to produce ascites containing high

30 concentrations of anti-TRAIL monoclonal antibodies. Alternatively, hybridoma cells
can be grown in vitro in flasks or roller bottles by various techniques. Monoclonal
antibodies produced in mouse ascites can be purified by ammonium sulfate
precipitation, followed by gel exclusion chromatography. Alternatively, affinity
chromatography based upon binding of antibody to protein A or protein G can be used,

35 as can affinity chromatography based upon binding to TRAIL.

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EXAMPLE 5: DNA Laddering Apootosis Assay

Human TRAIL was expressed and tested for the ability to induce apoptosis.
Oligonucleotides were synthesized that corresponded to the 3' and 5' ends of the coding
5 region of the human TRAIL gene, with Sail and NotI restriction sites incorporated at
the ends of the oligonucleotides. The coding region of the human TRAIL gene was
amplified by standard PCR techniques, using these oligonucleotides as primers. The
PCR reaction products were digested with the restriction endonucleases Sail and NotI,
then inserted into Sall/Notl-digested vector pDC409. pDC409 is an expression vector

10 for use in mammalian cells, but is also replicable in E. coli cells.

pDC409 is derived from an expression vector designated pDC406 (described in
McMahan et al., EMBO J. 10:2821, 1991, and in PCT application WO 91/18982,
hereby incorporated by reference). pDC406 contains origins of replication derived
from S V40, Epstein-Barr virus and pBR322 and is a derivative of HAV-EO described

15 by Dower et al., /. Immunol 142:4314 (1989). pDC406 differs from HAV-EO by the
deletion of an intron present in the adenovirus 2 tripartite leader sequence in HAV-EO.
DNA inserted into a multiple cloning site (containing a number of restriction
endonuclease cleavage sites) is transcribed and translated using regulatory elements
derived from HIV and adenovirus. The vector also contains a gene that confers

20 ampicillin resistance.

pDC409 differs from pDC406 in that a Bgl II site outside the mcs has been
deleted so that the mcs Bgl II site is unique. Two Pme 1 sites and one Srf 1 site have
been added to the mcs, and three stop codons (TAG) have been positioned downstream
of the mcs to function in all three reading frames. A T7 primer/promoter has been

25 added to aid in the DNA sequencing process.

The monkey kidney cell line CV-l/EBNA-1 (ATCC CRL 10478) was derived
by transfection of the C V- 1 cell line (ATCC CCL 70) with a gene encoding Epstein-
Bair virus nuclear antigen- 1 (EBNA-1) that constitutively expresses EBNA-1 driven
from the human CMV intermediate-early enhancer/promoter, as described by McMahan

30 et al., supra. The EBNA-1 gene allows for episomal replication of expression vectors,
such as pDC409, that contain the EBV origin of replication.

CV1/EBNA cells grown in Falcon T175 flasks were transfected with 15 |LLg of

either "empty" pDC409 or pDC409 containing the human TRAIL coding region. The
transformed cells were cultured for three days at 37°C and 10% CO2. The cells then
35 were washed with PBS, incubated for 20 minutes at 37°C in 50 mM EDTA, scraped off
of the flask with a cells scraper, and washed once in PBS. Next, the cells were fixed in
1% paraformaldehyde PBS for 10 minutes at 4°C, and washed 3x in PBS.

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Jurkat cells were used as the target cells in this assay, to determine whether the
TRAIL-expressing cells could induce apoptosis thereof. The Jurkat cell line, clone E6-
1, is a human acute T cell leukemia cell line available from the American Type Culture
Collection under accession no. ATCC TIB 152, and described in Weiss et al. (/.
5 Immunol 1 33: 123- 128, 1984). The Jurkat cells were cultured in RPMI media

supplemented with 10% fetal bovine serum and 10 Jig/ml streptomycin and penicillin to
a density of 200,000 to 500,000 cells per ml. Four million of these cells per well were
co-cultured in a 6 well plate with 2.5 mis of media with various combinations of fixed
cells, supernatants from cells transfected with Fas ligand, and various antibodies, as

10 indicated below.

After four hours the cells were washed once in PBS and pelleted at 1200 RPM
for 5 minutes in a desktop centrifuge. The pellets were resuspended and incubated for
ten minutes at 4°C in 500 (il of buffer consisting of 10 mM Tris-HCl, 10 mM EDTA,
pH 7.5, and 0.2% Triton X-100, which lyses the cells but leaves the nuclei intact. The

15 lysate was then spun at 4°C for ten minutes in a micro-centrifuge at 14,000 RPM. The
supernatants were removed and extracted three times with 1 ml of 25:24:1 phenol-
chloroform-isoamyl alcohol, followed by precipitation with NaOAC and ethanol in the
presence of 1 jig of glycogen carrier (Sigma).

The resulting pellets were resuspended in 10 mM Tris-HCl, 10 mM EDTA, pH

20 7.5 A and incubated with 10 Mg/ml RNase A at 37°C for 20 minutes. The DNA solutions
were then resolved by 1.5% agarose gel electrophoresis in Tris-Borate EDTA buffer,
stained with ethidium bromide and photographed while trans-illuminated with UV light.

The results were as follows. Fixed CV1/EBNA cells transfected with either
pDC409 or pDC409-TRAIL produced no detectable DNA laddering. pDC409-TRAIL

25 fixed cells co-cultured with Jurkat cells produced DNA laddering, but pDC409 fixed
cells co-cultured with Jurkat cells did not.

DNA laddering was also seen when Jurkat cells were co-cultured with
concentrated supernatants from COS cells transfected with DNA encoding human Fas
ligand in pDC409. The supernatants are believed to contain soluble Fas ligand that is

30 proteolytically released from the cell surface. The Fas ligand-induced DNA laddering
could be blocked by adding 10 jxg/ml of a soluble blocking monoclonal antibody
directed against Fas. This same antibody could not inhibit laddering of Jurkat DNA by
the pDC409-TRADL cells, which indicates that TRAIL does not induce apoptosis
through Fas.

35 In the same assay procedure, fixed CV1/EBNA cells transfected with pDC409-

TRAIL induced DNA laddering in U937 cells. U937 (ATCC CRL 1593) is a human

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histiocytic lymphoma cell line. The ratio of effector to target cells was 1 :4 (the same as
in the assay on Jurkat target cells).

The fragmentation of cellular DNA into a pattern known as DNA laddering is a
hallmark of apoptosis. In the foregoing assay, TRAIL induced apoptosis of a leukemia
5 cell line and a lymphoma cell line.

EXAMPLE 6: Northern Blot Analysis

Expression of TRAIL in a number of different tissue types was analysed in a
conventional northern blot procedure. Northern blots containing poly A + RNA from a

10 variety of adult human tissues (multiple tissue northern blots I and II) were obtained

from Clonetech (Palo Alto, CA). Other blots were prepared by resolving RNA samples
on a LI % agarose-formaldehyde gel, blotting onto Hybond-N as recommended by the
manufacturer (Amersham Corporation), and staining with methylene blue to monitor
RNA concentrations. The blots were probed with an antisense RNA riboprobe

15 corresponding to the entire coding region of human TRAIL.

Human TRAIL mRNA was detected in peripheral blood lymphocytes, colon,
small intestine, ovary, prostate, thymus, spleen, placenta, lung, kidney, heart,
pancreas, and skeletal muscle. TRAIL transcripts were found to be abundant in the
large cell anaplastic lymphoma cell line Kaipas 299 (Fischer et al., Blood, 72:234,

20 1988) and in tonsilar T cells. TRAIL message was present to a lesser degree in the
Burkitt lymphoma cell line designated Raji.

TRAIL mRNA was not detected in testis, brain, or liver, or in several T cell
lines. Little or no TRAIL transcripts were detected in freshly isolated peripheral blood
T cells (PBT), either unstimulated or stimulated with PMA and calcium ionophore for

25 20 hours.

EXAMPLE 7t Production of a Soluble TRAIL Polypeptide

A soluble human TRAIL polypeptide comprising amino acids 95 to 28 1 of SEQ
ID NO:2 was prepared as follows. This polypeptide is a fragment of the extracellular
30 domain, lacking the spacer region discussed above.

An expression vector encoding soluble human TRAIL was constructed by
fusing in-frame DNA encoding the following amino acid sequences (listed from N- to
C-terminus): a leader sequence derived from human cytomegalovirus (CMV), a
synthetic epitope designated Flag®, and amino acids 95-281 of human TRAIL. The
35 Flag® octapeptide (SEQ ID NO:7) facilitates purification of proteins fused thereto, as
described above and in Hopp et al. {Biotechnology 6:1204-1210, 1988).

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The TRADL-encoding DNA fragment was isolated and amplified by polymerase
chain reaction (PCR), using oligonucleotide primers that defined the termini of a DNA
fragment encoding amino acids 95-281 of SEQ ID NO:2. The 3' primer was a 31-mer
that additionally added a NotI site downstream of the TRADL-encoding sequence. The
5 5' primer added an Spel site and a Flag® epitope encoding sequence upstream of the
TRAIL-encoding sequence. PCR was conducted by conventional procedures, using
the above-described human TRAIL cDNA as the template.

The reaction products were digested with Spel and NotI, and inserted into the
expression vector pDC409 (described in example 5), which had been cleaved with Sail

10 and NotI. Annealed oligonucleotides that form a Sall-Spel fragment encoding a CMV
open reading frame leader were also ligated into the vector. The amino acid sequence of
the CMV-derived leader is presented as SEQ ID NO:9. Amino acids 1 to 29 of SEQ ID
NO:9 are encoded by CMV DNA, whereas amino acids 30 to 32 are encoded by
oligonucleotides employed in constructing the vector. E. coli cells were transfected

15 with the ligation mixture, and the desired recombinant expression vector was isolated
therefrom.

CV1-EBNA cells (ATCC CRL 10478; described in example 5) were transfected
with the recombinant vector, which is designated pDC409-Flag-shTRAIL, and cultured
to allow expression and secretion of the soluble Flag®-TRAIL polypeptide. Culture

20 supernatants were harvested 3 days after transfection and applied to a column

containing an anti-Flag® antibody designated M2 immobilized on a solid support. The
column then was washed with PBS. The monoclonal antibody M2 is described in
Hopp et al., supra, and available from Kodak Scientific Imaging Systems, New Haven,
Connecticut. 800m1 fractions were eluted from the column with 50 mM citrate, and

25 immediately neutralized in 0.45 ml 1M Tris (pH 8). Fractions were adjusted to 10%
glycerol and stored at -20°C until needed.

This soluble recombinant Flag®/human TRAIL expressed in CV1/EBNA cells
has an apparent molecular weight of 28 kD when analyzed by SDS-polyacrylamide gel
electrophoresis (SDS-PAGE). The Flag® moiety contributes an estimated 880 daltons

30 to the total molecular weight. Gel filtration analysis of purified soluble Flag®/TRAIL
suggests that the molecule is multimeric in solution with a size of -80 kD. While not
wishing to be bound by theory, the gel filtration analysis suggests that the soluble
recombinant Flag®/human TRAIL naturally formed a combination of dimers and
trimers, with trimers predominating.

35 An expression vector designated pDC409-Flag-smTRAIL, which encodes a

CMV leader-Flag®- soluble murine TRAIL protein, was constructed by analogous
procedures. A DNA fragment encoding a soluble murine TRAIL polypeptide was

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isolated and amplified by PCR. Oligonucleotides that defined the termini of DNA
encoding amino acids 99 to 291 of the murine TRAIL sequence of SEQ ID NO: 6 were
employed as the 5' and 3 1 primers in the PCR.

5 EXAMELE »= LSSIS of Leukemia Cells hv Soluble TRAIL

In example 5, cells expressing human TRAIL induced apoptosis of Jurkat cells
(a leukemia cell line). In the following study, a soluble human TRAIL polypeptide
killed Jurkat cells.

Jurkat cells were cultured to a density of 200,000 to 500,000 cells per ml in
10 RPMI medium supplemented with 10% fetal bovine serum, 100 pg/ml streptomycin,
and 100 jig/ml penicillin. The cells (in 96- well plates at 50,000 cells per well in a
volume of 100 jol) were incubated for twenty hours with the reagents indicated in Figure
1. "TRAIL supe." refers to conditioned supernatant (10 pi per well) from CV1/EBNA
cells transfected with pDC409-Flag-shTRAIL (see example 7). "Control supe." refers
15 to supernatant from CV1/EBNA cells transfected with empty vector. Where indicated,
immobilized anti-Flag® antibody M2 ("Imm. M2") was added at a concentration of 10
|ig/ml in a volume of 1 00 |il per well and allowed to adhere either overnight at 4°C or

for 2 hours at 37°C, after which wells were aspirated and washed twice with PBS to
remove unbound antibody. Jurkat cells treated with Fas ligand or M3, a blocking

20 monoclonal antibody directed against Fas, (Alderson et al., J. Exp. Med. 181:71, 1995;
and PCT application WO 95/10540) were included in the assay as indicated.

Metabolic activity of the thus-treated cells was assayed by metabolic conversion
of alamar Blue dye, in the following procedure. Alamar Blue conversion was measured
by adding 10 \A of alamar Blue dye (Biosource International, Camarillo, CA) per well,

25 and subtracting the optical density (OD) at 550-600 nm at the time the dye was added

from the OD 550-600 nm after four hours. No conversion of dye is plotted as 0 percent
viability, and the level of dye conversion in the absence of TRAIL is plotted as 100
percent viability. Percent viability was calculated by multiplying the ratio of staining of
experimental versus control cultures by 100.

30 The results are presented in Figure 1 . Error bars represent the standard

deviation of measurements from four independent wells, and the values are the average
of these measurements.

The TRAIL-containing supernatant caused a significant reduction in viability of
Jurkat cells. A greater reduction of cell viability resulted from contact with a

35 combination of TRAIL-containing supernatant and immobilized anti-Flag® antibody
M2. One possible explanation is that M2 facilitates cross-linking of the Flag®/TRAIL-
receptor complexes, thereby increasing the strength of signaling.

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Fas ligand demonstrated the ability to kill Jurkat cells. The anti-Fas antibody
M3 inhibited the activity of Fas ligand, but not the activity of TRAIL.

In order to confirm that the changes in dye conversion in the alamar Blue assay
were due to cell death, the decrease in cell viability induced by TRAIL was confirmed
5 by staining the cells with trypan blue.

EXAMPLE 9: Lvsis of Leukemia and Lymphoma Cells

In examples 5 and 8, TRAIL induced apoptosis of a leukemia cell line (Jurkat)
and a lymphoma cell line (U937). The following study further demonstrates the ability
10 of TRAIL to kill leukemia and lymphoma cells.

The human cell lines indicated in Table I were cultured to a density of 200,000
to 500,000 cells per ml in RPMI medium supplemented with 10% fetal bovine serum,
100 |ig/ml streptomycin, and 100 M.g/ml penicillin. The cells (in 96-well plates at

50,000 cells per well in a volume of 100 pi) were incubated for twenty hours with
15 conditioned supernatants (10 jul per well) from pDC409-Flag-shTRAIL transfected
GV1/EBNA cells.

Metabolic activity was assayed by conversion of alamar Blue dye, in the assay
procedure described in example 8. The results are presented in Table I.

In order to confirm that the changes in dye conversion in the alamar Blue assay
20 were due to cell death, the decrease in cell viability induced by TRAIL was confirmed
by staining the cells with trypan blue. Crystal violet staining, performed as described
by Flick and Gifford (/. Immunol. Methods 68:167-175, 1984), also confirmed the
results seen in the alamar Blue assay. The apoptotic nature of the cell death was
confirmed by trypan blue staining and visualization of apoptotic fragmentation by
25 microscopy.

As shown in Table I, many cancer cell lines were sensitive to TRAIL mediated
killing. The susceptibility of additional cell types to TRAIL mediated apoptosis can be
determined using the assay procedures described in this examples section.

TRAIL exhibited no significant cytotoxic effect on the cell lines THP-1 , K562,

30 Karpas 299, and MP-1. K299, also known as Karpas 299, (DSM-ACC31) was
established from peripheral blood of a male diagnosed with high grade large cell
anaplastic lymphoma (Fischer et al., Blood, 72:234, 1988). MP-1 is a spontaneously
derived EBV-transformed B cell line (Goodwin et al., Cell 13:441, 1993). While not
wishing to be bound by theory, it is possible that these four cell lines do not express a

35 receptor for TRAIL, or are characterized by upregulation of a gene that inhibits
apoptosis.

WO 97/01633 PCT/US96/10895

Table 1. Effect of soluble TRAIL on cell line viability

Cell Line

Description

Percent Viability a

Bjab

Burkitt lymphoma

0.5 + 3.8

Ramos

Burkitt lymphoma

12.1 + 2.1

U937

histiocytic lymphoma

25.2 + 8.2

HL60

promyelocytic leukemia

59.5 + 3.2

Raji

Burkitt lymphoma

64.9 + 4.5

Daudi

Burkitt lymphoma

70.2 + 4.2

THP-1

monocytic cell line

92.3 + 6.8

K562

chronic myelogenous leukemia

97.1 + 4.8

K299

large cell anaplastic lymphoma

99.0 + 4.3

MP-1

spontaneous B cell line

104.9 + 11.7

a Results are means ± SEMs of 4 wells for each data point

EXAMPLE 10: Cross-Species Activity of TRATL

Interspecies cross-reactivity of human and murine TRAIL was tested as
follows. Murine and human TRAIL were incubated with the human melanoma cell line
A375 (ATCC CRL 1619). Since this is an adherent cell line, a crystal violet assay,

10 rather than alamar Blue, was used to determine cell viability. A375 cells were cultured
in DMEM supplemented with 10% fetal bovine serum, 100 |ig/ml streptomycin, and
100 \xg/m\ penicillin. The cells (in 96- well plates at 10,000 cells per well in a volume of
100 jjI) were incubated for 72 hours with the soluble murine TRAIL described in
example 7. Crystal violet staining was performed as described by (Flick and Gifford

15 (/. Immunol. Methods 68:167-175, 1984). The results demonstrated that both human
and murine TRAIL are active on these human cells, in that murine and human TRAIL
killed A375 cells.

The ability of human TRAIL to act on murine cells was tested, using the
immortalized murine fibroblast cell line L929. Incubation of L929 cells with either
20 human or murine TRAIL resulted in a decrease in crystal violet staining, thus

demonstrating that human and murine TRAIL are active on (induced apoptosis of)
murine cells. In addition to crystal violet, cell death was confirmed by trypan-blue
staining.

25 EXAMPLE 11: Lvsis of CMV-Tnfected Cells

The following experiment demonstrates that the soluble Flag®-human TRAIL
protein prepared in example 7 has a cytotoxic effect on virally infected cells.

Normal human gingival fibroblasts were grown to confluency on 24 well plates
in 10% CO2 and DMEM medium supplemented with 10% fetal bovine serum, 100

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ixg/ml streptomycin, and 100 pig/ml penicillin. Samples of the fibroblasts were treated
as indicated in Figure 2. Concentrations of cytokines were 10 ng/ml for y-interferon

and 30 ng/ml of soluble Flag®-human TRAIL. All samples receiving TRAIL also
received a two-fold excess by weight of anti-Flag® antibody M2 (described above),
5 which enhances TRAIL activity (presumably by crosslinking).

Pretreatment of cells with the indicated cytokines was for 20 hours. To infect
cells with cytomegalovirus (CMV), culture media were aspirated and the cells were
infected with CMV in DMEM with an approximate MOI (multiplicity of infection) of 5.
After two hours the virus containing media was replaced with DMEM and cytokines

10 added as indicated. After 24 hours the cells were stained with crystal violet dye as
described (Flick and Gifford, 1984, supra). Stained cells were washed twice with
water, disrupted in 200 yl of 2% sodium deoxycholate, diluted 5 fold in water, and the
OD taken at 570 nm. Percent maximal staining was calculated by normalizing ODs to
the sample that showed the greatest staining. Similar results were obtained from several

15 independent experiments.

The results presented in Figure 2 demonstrate that TRAIL specifically killed
CMV infected fibroblasts. This cell death was enhanced by pretreatment of the cells
with y-interferon. No significant death of non-virally infected fibroblasts resulted from
contact with TRAIL.

EXAMPLE 12: Assay to Identify Blocking Antihnriips

Blocking antibodies directed against TRAIL may be identified by testing
antibodies for the ability to inhibit a particular biological activity of TRAIL. In the
following assay, a monoclonal antibody was tested for the ability to inhibit TRAIL-

25 mediated apoptosis of Jurkat cells. The Jurkat cell line is described in example 5.

A hybridoma cell line producing a monoclonal antibody raised against a
Hag®/soluble human TRAIL fusion protein was employed in the assay. Supernatants
from the hybridoma cultures were incubated with 20 ng/ml Flag®/soluble human
TRAIL crosslinked with 40 ng/ml anti-Flag® monoclonal antibody M2, in RPMI

30 complete media in a 96 well microtiter plate. An equivalent amount of fresh hybridoma
culture medium was added to control cultures. The Flag®/soluble human TRAIL
fusion protein and the monoclonal antibody designated M2 are described in example 7.

The hybridoma supernatant was employed at a 1 :50 (v/v) dilution (starting
concentration), and at two fold serial dilutions thereof. After incubation at 37°C, 10%

35 C0 2 , for 30 minutes, 50,000 Jurkat cells were added per well, and incubation was
continued for 20 hours.

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Cell viability was then assessed measuring metabolic conversion of alamar blue
dye. An alamar blue conversion assay procedure is described in example 8. The
monoclonal antibody was found to inhibit the apoptosis of Jurkat cells induced by
Hag®/soluble human TRAIL.

EXAMPLE 13; TRAIL Blocking Study

Human microvascular endothelial cells of dermal origin were treated for 16-18
hours with plasma from patients with thrombotic thrombocytopenic purpura (TIP) or
with control plasma, either alone or in the presence of anti-TRACL polyclonal
antiserum. A 1:2000 dilution of the antiserum was employed. The plasma was from
two TIP patients, designated #1 and #2 below, The cells employed in the assays were
MVEC-1 (HMVEC 2753, purchased from Clonetics, San Diego, CA) and MVEC-2
(DHMVEC 30282, purchased from Cell Systems, Kirkland, WA). Cultures of these
cells can be maintained as described in Laurence et al. (Blood, 87:3245, 1996).

The results were as follows. The data shown are from DNA histograms of cells
stained with propidium iodide, and "A 0 peak" represents the apoptotic peak (see Oyaizu
et al., Blood, 82:3392, 1993; Nicoletti et al., /. Immunol Methods, 139:271, 1991;
and Laurence et al., Blood, 75:696, 1990).

Experiment 1

25 Experiment 2

30 Experiment 3

Experiment 4

Microvascular EC

Plasma (1 %)

Anu'bodv

% A fl peak

Dermal MVEC-1

control

Dermal MVEC-1

TTP(#1)

19.5

Dermal MVEC-1

TTP (#1)

0.3

Dermal MVEC-2

control

Dermal MVEC-2

TTP (#2)

20.0

Dermal MVEC-2

TTP (#2)

control Ab

13.1

Dermal MVEC-2

TTP (#2)

0.2

Dermal MVEC-1

TTP (#1)

50.1

Dermal MVEC-1

TTP (#1)

10.6

Dermal MVEC-2

control

Dermal MVEC-2

TTP (#1)

13.9

Dermal MVEC-2

TTP (#1)

control Ab

14.1

Dermal MVEC-2

TTP (#1)

0.6

The data reveal that plasma derived from TTP patients induces apoptosis of
microvascular endothelial cells of dermal origin. This apoptosis was inhibited by
polyclonal antibodies directed against TRAIL.

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SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: Immunex Corporation,
(ii) TITLE OF INVENTION: Cytokine That Induces Apoptosis
(iii) NUMBER OF SEQUENCES: 9

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Kathryn A. Anderson, Immunex Corporation

(B) STREET: 51 University Street

(D) STATE: WA

(E) COUNTRY: USA

(F) ZIP: 98101

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: Apple Macintosh

(D) SOFTWARE: Microsoft Word, Version 6.0.1

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: — to be assigned —

(B) FILING DATE: 25-JUN-1996

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: US 08/496,632

(B) FILING DATE: 2 9-JUN-1995

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: US 08/548,368

(B) FILING DATE: 01-NOV-1995

(viii) ATTORNEY/ AGENT INFORMATION:

(A) NAME: Anderson, Kathryn A.

(B) REGISTRATION NUMBER : 32,172

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (206) 587-0430

(B) TELEFAX: (206) 233-0644

(2) INFORMATION FOR SEQ ID NO : 1 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1751 base pairs

(B) TYPE: nucleic acid

(D) TOPOLOGY: linear

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(ii) MOLECULE TYPE: cDNA to mRNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO

(vii) IMMEDIATE SOURCE:
(B) CLONE: huAIC

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 8 8 .. 933

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 1 :

CCTCACTGAC TATAAAAGAA TAGAGAAGGA AGGGCTTCAG TGACCGGCTG CCTGGCTGAC 60

TTACAGCAGT CAGACTCTGA CAGGATC ATG GCT ATG ATG GAG GTC CAG GGG 111

Met Ala Met Met Glu Val Gin Gly

1 5

GGA CCC AGC CTG GGA CAG ACC TGC GTG CTG ATC GTG ATC TTC ACA GTG 159
Gly Pro Ser Leu Gly Gin Thr Cys Val Leu lie Val lie Phe Thr Val
10 15 20

CTC CTG CAG TCT CTC TGT GTG GCT GTA ACT TAC GTG TAC TTT ACC AAC 207
Leu Leu Gin Ser Leu Cys Val Ala Val Thr Tyr Val Tyr Phe Thr Asn
25 30 35 40

GAG CTG AAG CAG ATG CAG GAC AAG TAC TCC AAA AGT GGC ATT GCT TGT 255
Glu Leu Lys Gin Met Gin Asp Lys Tyr Ser Lys Ser Gly lie Ala Cys

45 50 55

TTC TTA AAA GAA GAT GAC AGT TAT TGG GAC CCC AAT GAC GAA GAG AGT 303
Phe Leu Lys Glu Asp Asp Ser Tyr Trp Asp Pro Asn Asp Glu Glu Ser

60 65 70

ATG AAC AGC CCC TGC TGG CAA GTC AAG TGG CAA CTC CGT CAG CTC GTT 351
Met Asn Ser Pro Cys Trp Gin Val Lys Trp Gin Leu Arg Gin Leu Val

75 80 85

AGA AAG ATG ATT TTG AGA ACC TCT GAG GAA ACC ATT TCT ACA GTT CAA 399
Arg Lys Met lie Leu Arg Thr Ser Glu Glu Thr lie Ser Thr Val Gin
90 95 100

GAA AAG CAA CAA AAT ATT TCT CCC CTA GTG AGA GAA AGA GGT CCT CAG 447
Glu Lys Gin Gin Asn lie Ser Pro Leu Val Arg Glu Arg Gly Pro Gin
105 110 115 120

AGA GTA GCA GCT CAC ATA ACT GGG ACC AGA GGA AGA AGC AAC ACA TTG 495
Arg Val Ala Ala His lie Thr Gly Thr Arg Gly Arg Ser Asn Thr Leu

125 130 135

TCT TCT CCA AAC TCC AAG AAT GAA AAG GCT CTG GGC CGC AAA ATA AAC 543
Ser Ser Pro Asn Ser Lys Asn Glu Lys Ala Leu Gly Arg Lys lie Asn

140 145 150

TCC TGG GAA TCA TCA AGG AGT GGG CAT TCA TTC CTG AGC AAC TTG CAC 591

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Ser Trp Glu Ser Ser Arg Ser Gly His Ser Phe Leu Ser Asn Leu His
155 160 165

TTG AGG AAT GGT GAA CTG GTC ATC CAT GAA AAA GGG TTT TAC TAC ATC 639
Leu Arg Asn Gly Glu Leu Val lie His Glu Lys Gly Phe Tyr Tyr lie
170 175 180

TAT TCC CAA ACA TAC TTT CGA TTT CAG GAG GAA ATA AAA GAA AAC ACA 687
Tyr Ser Gin Thr Tyr Phe Arg Phe Gin Glu Glu lie Lys Glu Asn Thr
185 190 195 200

AAG AAC GAC AAA CAA ATG GTC CAA TAT ATT TAC AAA TAC ACA AGT TAT 735
Lys Asn Asp Lys Gin Met Val Gin Tyr lie Tyr Lys Tyr Thr Ser Tyr

205 210 215

CCT GAC CCT ATA TTG TTG ATG AAA AGT GCT AGA AAT AGT TGT TGG TCT 783
Pro Asp Pro lie Leu Leu Met Lys Ser Ala Arg Asn Ser Cys Trp Ser

220 225 230

AAA GAT GCA GAA TAT GGA CTC TAT TCC ATC TAT CAA GGG GGA ATA TTT 831
Lys Asp Ala Glu Tyr Gly Leu Tyr Ser lie Tyr Gin Gly Gly lie Phe
235 240 245

GAG CTT AAG GAA AAT GAC AGA ATT TTT GTT TCT GTA ACA AAT GAG CAC 87 9

Glu Leu Lys Glu Asn Asp Arg lie Phe Val Ser Val Thr Asn Glu His
250 255 260

TTG ATA GAC ATG GAC CAT GAA GCC AGT TTT TTC GGG GCC TTT TTA GTT 927
Leu lie Asp Met Asp His Glu Ala Ser Phe Phe Gly Ala Phe Leu Val
265 270 275 280

GGC TAA CTGACCTGGA AAGAAAAAGC AATAACCTCA AAGTGACTAT TCAGTTTTCA 983
Gly *

GG AT GAT AC A CTATGAAGAT GTTTCAAAAA ATCTGACCAA AACAAACAAA CAGAAAACAG 1043
AAAACAAAAA AACCTCTATG CAATCTGAGT AGAGCAGCCA CAACCAAAAA ATTCTACAAC 1103
ACACACTGTT CTGAAAGTGA CTCACTTATC CCAAGAAAAT GAAATTGCTG AAAGATCTTT 1163
CAGGACTCTA CCTCATATCA GTTTGCTAGC AGAAATCTAG AAGACTGTCA GCTTCCAAAC 1223
ATTAATGCAA T GGT T AAC AT CTTCTGTCTT TATAATCTAC TCCTTGTAAA GACTGTAGAA 1283
GAAAGCGCAA CAATCCATCT CTCAAGTAGT GTATCACAGT AGTAGCCTCC AGGTTTCCTT 1343
AAGGGACAAC ATCCTTAAGT CAAAAGAGAG AAGAGGCACC ACTAAAAGAT CGCAGTTTGC 1403
CTGGTGCAGT GGCTCACACC TGTAATCCCA ACATTTTGGG AACCCAAGGT GGGTAGATCA 14 63
CGAGATCAAG AG ATC AAG AC CATAGTGACC AAC AT AGT GA AACCCCATCT CTACTGAAAG 1523
TGCAAAAATT AGCTGGGTGT GTTGGCACAT GCCTGTAGTC CCAGCTACTT GAGAGGCTGA 1583
GGCAGGAGAA TCGTTTGAAC CCGGGAGGCA GAGGTTGCAG TGT GGT GAGA TCATGCCACT 1643
ACACTCCAGC CT GGC GAC AG AGCGAGACTT GGTTTCAAAA AAAAAAAAAA AAAAAAACTT 1703
CAGTAAGTAC GTGTTATTTT TTTCAATAAA ATTCTATTAC AGTATGTC 1751

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(2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 281 amino acids

(B) TYPE: amino acid
(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

Met Ala Met Met Glu Val Gin Gly Gly Pro Ser Leu Gly Gin Thr Cys
15 10 15

Val Leu lie Val lie Phe Thr Val Leu Leu Gin Ser Leu Cys Val Ala

20 25 30

Val Thr Tyr Val Tyr Phe Thr Asn Glu Leu Lys Gin Met Gin Asp Lys
35 40 45

Tyr Ser Lys Ser Gly lie Ala Cys Phe Leu Lys Glu Asp Asp Ser Tyr
50 55 60

Trp Asp Pro Asn Asp Glu Glu Ser Met Asn Ser Pro Cys Trp Gin Val
65 70 75 80

Lys Trp Gin Leu Arg Gin Leu Val Arg Lys Met lie Leu Arg Thr Ser

85 90 95

Glu Glu Thr lie Ser Thr Val Gin Glu Lys Gin Gin Asn lie Ser Pro

100 105 110

Leu Val Arg Glu Arg Gly Pro Gin Arg Val Ala Ala His lie Thr Gly
115 120 125

Thr Arg Gly Arg Ser Asn Thr Leu Ser Ser Pro Asn Ser Lys Asn Glu
130 135 140

Lys Ala Leu Gly Arg Lys lie Asn Ser Trp Glu Ser Ser Arg Ser Gly
145 150 155 160

His Ser Phe Leu Ser Asn Leu His Leu Arg Asn Gly Glu Leu Val lie

165 170 175

His Glu Lys Gly Phe Tyr Tyr lie Tyr Ser Gin Thr Tyr Phe Arg Phe

180 185 190 •

Gin Glu Glu lie Lys Glu Asn Thr Lys Asn Asp Lys Gin Met Val Gin
195 200 205

Tyr lie Tyr Lys Tyr Thr Ser Tyr Pro Asp Pro lie Leu Leu Met Lys
210 215 220

Ser Ala Arg Asn Ser Cys Trp Ser Lys Asp Ala Glu Tyr Gly Leu Tyr
225 230 235 240

Ser lie Tyr Gin Gly Gly lie Phe Glu Leu Lys Glu Asn Asp Arg lie

245 250 255

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PCT/US96/10895

Phe Val Ser Val Thr Asn Glu His Leu lie Asp Met Asp His Glu Ala

260 265 270

Ser Phe Phe Gly Ala Phe Leu Val Gly *
275 280

(2) INFORMATION FOR SEQ ID NO: 3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1521 base pairs

(B) TYPE: nucleic acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA to mRNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO

(vii) IMMEDIATE SOURCE:

(B) CLONE: HuAIC-dv

<ix) FEATURE:

(A) NAME /KEY: CDS

(B) LOCATION: 7 8.. 383

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 3 :

AATTCCGGAA TAGAGAAGGA AGGGCTTCAG TGACCGGCTG CCTGGCTGAC TTACAGCAGT 60

CAGACTCTGA CAGGATC ATG GCT ATG ATG GAG GTC CAG GGG GGA CCC AGC 110

Met Ala Met Met Glu Val Gin Gly Gly Pro Ser
15 10

CTG GGA CAG ACC TGC GTG CTG ATC GTG ATC TTC ACA GTG CTC CTG CAG 158
Leu Gly Gin Thr Cys Val Leu He Val He Phe Thr Val Leu Leu Gin

15 20 25

TCT CTC TGT GTG GCT GTA ACT TAC GTG TAC TTT ACC AAC GAG CTG AAG 20 6
Ser Leu Cys Val Ala Val Thr Tyr Val Tyr Phe Thr Asn Glu Leu Lys
30 35 40

CAG ATG CAG GAC AAG TAC TCC AAA AGT GGC ATT GCT TGT TTC TTA AAA 254
Gin Met Gin Asp Lys Tyr Ser Lys Ser Gly He Ala Cys Phe Leu Lys
45 50 55

GAA GAT GAC AGT TAT TGG GAC CCC AAT GAC GAA GAG AGT ATG AAC AGC 302
Glu Asp Asp Ser Tyr Trp Asp Pro Asn Asp Glu Glu Ser Met Asn Ser
60 65 70 75

CCC TGC TGG CAA GTC AAG TGG CAA CTC CGT CAG CTC GTT AGA AAG ACT 350
Pro Cys Trp Gin Val Lys Trp Gin Leu Arg Gin Leu Val Arg Lys Thr

80 85 90

CCA AGA ATG AAA AGG CTC TGG GCC GCA AAA TAA ACTCCTGGGA ATCATCAAGG 403
Pro Arg Met Lys Arg Leu Trp Ala Ala Lys *

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PCT/US96/10895

95 100

AGTGGGCATT CATTCCTGAG CAACTTGCAC TTGAGGAATG GTGAACTGGT CATCCATGAA 463

AAAGGGTTTT ACTACATCTA TTCCCAAACA TACTTTCGAT TTCAGGAGGA AATAAAAGAA 523

AACACAAAGA ACGACAAACA AATGGTCCAA TATATTTACA AATACACAAG TTATCCTGAC 583

CCTATATTGT TGATGAAAAG TGCTAGAAAT AGTTGTTGGT CTAAAGATGC AGAATATGGA 643

CTCTATTCCA TCTATCAAGG GGGAATATTT GAGCTTAAGG AAAAT G AC AG AATTTTTGTT 703

TCTGTAACAA ATGAGCACTT GATAGACATG GACCATGAAG CCAGTTTTTT CGGGGCCTTT 763

TTAGTTGGCT AACTGACCTG GAAAGAAAAA GCAATAACCT CAAAGTGACT ATTCAGTTTT 823

CAGGATGATA CACTATGAAG ATGTTTCAAA AAATCTGACC AAAACAAACA AACAGAAAAC 883

AGAAAACAAA AAAACCTCTA TGCAATCTGA GTAGAGCAGC CACAACCAAA AAATTCTACA 943

ACACACACTG TTCTGAAAGT GACTCACTTA TCCCAAGAGA ATGAAATTGC TGAAAGATCT 1003

TTCAGGACTC TACCTCATAT CAGTTTGCTA GCAGAAATCT AGAAGACTGT CAGCTTCCAA 1063

ACATTAATGC AGTGGTTAAC ATCTTCTGTC TTTATAATCT ACTCCTTGTA AAGACTGTAG 1123

AAGAAAGCGC AACAATCCAT CTCTCAAGTA GTGTATCACA GTAGTAGCCT CCAGGTTTCC 1183

T T AAGGGAC A ACATCCTTAA GTCAAAAGAG AGAAGAGGCA CCACTAAAAG ATCGCAGTTT 1243

GCCTGGTGCA GTGGCTCACA CCTGTAATCC CAACATTTTG GGAACCCAAG GTGGGTAGAT 1303

CACGAGATCA AGAGATCAAG ACCATAGTGA CCAACATAGT GAAACCCCAT CTCTACTGAA 1363

AGTGCAAAAA TTAGCTGGGT GTGTTGGCAC ATGCCTGTAG TCCCAGCTAC TTGAGAGGCT 1423

GAGGCAGGAG AATCGTTTGA ACCCGGGAGG CAGAGGTTGC AGTGTGGTGA GATCATGCCA 1483

CTACACTCCA GCCTGGCGAC AGAGCGAGAC TTGGTTTC 1521

(2) INFORMATION FOR SEQ ID NO: 4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 101 amino acids

(B) TYPE: amino acid
(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 4 :

Met Ala Met Met Glu Val Gin Gly Gly Pro Ser Leu Gly Gin Thr Cys
15 10 15

Val Leu lie Val lie Phe Thr Val Leu Leu Gin Ser Leu Cys Val Ala

20 25 30

Val Thr Tyr Val Tyr Phe Thr Asn Glu Leu Lys Gin Met Gin Asp Lys
35 40 45

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Tyr Ser Lys Ser Gly lie Ala Cys Phe Leu Lys Glu Asp Asp Ser Tyr
50 55 60

Trp Asp Pro Asn Asp Glu Glu Ser Met Asn Ser Pro Cys Trp Gin Val
65 70 75 80

Lys Trp Gin Leu Arg Gin Leu Val Arg Lys Thr Pro Arg Met Lys Arg

85 90 95

Leu Trp Ala Ala Lys *

100

(2) INFORMATION FOR SEQ ID NO : 5 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 13 66 base pairs

(B) TYPE: nucleic acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA to mRNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO

(vii) IMMEDIATE SOURCE:
(B) CLONE: MuAIC

(ix) FEATURE:

(A) NAME /KEY : CDS

(B) LOCATION: 47.. 919

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 5 :

TGCTGGGCTG CAAGTCTGCA TTGGGAAGTC AGACC TGGAC AGCAGT ATG CCT TCC 55

Met Pro Ser
1

TCA GGG GCC CTG AAG GAC CTC AGC TTC AGT CAG CAC TTC AGG ATG ATG 103
Ser Gly Ala Leu Lys Asp Leu Ser Phe Ser Gin His Phe Arg Met Met
5 10 15

GTG ATT TGC ATA GTG CTC CTG CAG GTG CTC CTG CAG GCT GTG TCT GTG 151
Val lie Cys lie Val Leu Leu Gin Val Leu Leu Gin Ala Val Ser Val
20 25 30 35

GCT GTG ACT TAC ATG TAC TTC ACC AAC GAG ATG AAG CAG CTG CAG GAC 199
Ala Val Thr Tyr Met Tyr Phe Thr Asn Glu Met Lys Gin Leu Gin Asp

40 45 50

AAT TAC TCC AAA ATT GGA CTA GCT TGC TTC TCA AAG ACG GAT GAG GAT 247
Asn Tyr Ser Lys lie Gly Leu Ala Cys Phe Ser Lys Thr Asp Glu Asp

55 60 65

TTC TGG GAC TCC ACT GAT GGA GAG ATC TTG AAC AGA CCC TGC TTG CAG 2 95
Phe Trp Asp Ser Thr Asp Gly Glu lie Leu Asn Arg Pro Cys Leu Gin

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70 75 80

GTT AAG AGG CAA CTG TAT CAG CTC ATT GAA GAG GTG ACT TTG AGA ACC 343
Val Lys Arg Gin Leu Tyr Gin Leu lie Glu Glu Val Thr Leu Arg Thr
85 90 95

TTT CAG GAC ACC ATT TCT ACA GTT CCA GAA AAG CAG CTA AGT ACT CCT 391
Phe Gin Asp Thr lie Ser Thr Val Pro Glu Lys Gin Leu Ser Thr Pro
100 105 110 115

CCC TTG CCC AGA GGT GGA AGA CCT CAG AAA GTG GCA GCT CAC ATT ACT 439
Pro Leu Pro Arg Gly Gly Arg Pro Gin Lys Val Ala Ala His lie Thr

120 125 130

GGG ATC ACT CGG AGA AGC AAC TCA GCT TTA ATT CCA ATC TCC AAG GAT 487
Gly lie Thr Arg Arg Ser Asn Ser Ala Leu lie Pro lie Ser Lys Asp

135 140 145

GGA AAG ACC TTA GGC CAG AAG ATT GAA TCC TGG GAG TCC TCT CGG AAA 535
Gly Lys Thr Leu Gly Gin Lys lie Glu Ser Trp Glu Ser Ser Arg Lys

150 155 160 265

GGG CAT TCA TTT CTC AAC CAC GTG CTC TTT AGG AAT GGA GAG CTG GTC 583
Gly His Ser Phe Leu Asn His Val Leu Phe Arg Asn Gly Glu Leu Val
165 170 175

ATC GAG CAG GAG GGC CTG TAT TAC ATC TAT TCC CAA ACA TAC TTC CGA 631
lie Glu Gin Glu Gly Leu Tyr Tyr lie Tyr Ser Gin Thr Tyr Phe Arg
180 185 190 195

TTT CAG GAA GCT GAA GAC GCT TCC AAG ATG GTC TCA AAG GAC AAG GTG 67 9
Phe Gin Glu Ala Glu Asp Ala Ser Lys Met Val Ser Lys Asp Lys Val

200 205 210

AGA ACC AAA CAG CTG GTG CAG TAC ATC TAC AAG TAC ACC AGC TAT CCG 727
Arg Thr Lys Gin Leu Val Gin Tyr lie Tyr Lys Tyr Thr Ser Tyr Pro

215 220 225

GAT CCC ATA GTG CTC ATG AAG AGC GCC AGA AAC AGC TGT TGG TCC AGA 775
Asp Pro lie Val Leu Met Lys Ser Ala Arg Asn Ser Cys Trp Ser Arg
230 235 240

GAT GCC GAG TAC GGA CTG TAC TCC ATC TAT CAG GGA GGA TTG TTC GAG 823
Asp Ala Glu Tyr Gly Leu Tyr Ser lie Tyr Gin Gly Gly Leu Phe Glu
245 250 255

CTA AAA AAA AAT GAC AGG ATT TTT GTT TCT GTG ACA AAT GAA CAT TTG 871
Leu Lys Lys Asn Asp Arg lie Phe Val Ser Val Thr Asn Glu His Leu
260 265 270 275

ATG GAC CTG GAT CAA GAA GCC AGC TTC TTT GGA GCC TTT TTA ATT AAC 919
Met Asp Leu Asp Gin Glu Ala Ser Phe Phe Gly Ala Phe Leu lie Asn

280 285 290

TAAATGACCA GTAAAGATCA AACACAGCCC TAAAGTACCC AGTAATCTTC T AGG TTG AAG 97 9

GCATGCCTGG AAAGCGACTG AACTGGTTAG GATATGGCCT GGCTGTAGAA ACCTCAGGAC 103 9

AGATGTGACA GAAAGGCAGC TGGAACTCAG CAGCGACAGG CCAACAGTCC AGCCACAGAC 1099

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ACTTTCGGTG TTTCATCGAG AGACTTGCTT TCTTTCCGCA AAATGAGATC ACTGTAGCCT 1159
TTCAATGATC TACCTGGTAT CAGTTTGCAG AGATCTAGAA GACGTCCAGT TTCTAAATAT 1219
TTATGCAACA ATTGACAATT TTCACCTTTG TTATCTGGTC CAGGGGTGTA AAGCCAAGTG 127 9
CTCACAAGCT GTGTGCAGAC CAGGATAGCT ATGAATGCAG GTCAGCATAA AAATCACAGA 1339
ATATCTCACC TACTAAAAAA AAAAAAA 1366

(2) INFORMATION FOR SEQ ID NO: 6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2 91 amino acids

(B) TYPE: amino acid
(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 6 :

Met Pro Ser Ser Gly Ala Leu Lys Asp Leu Ser Phe Ser Gin His Phe
15 10 15

Arg Met Met Val lie Cys lie Val Leu Leu Gin Val Leu Leu Gin Ala

20 25 30

Val Ser Val Ala Val Thr Tyr Met Tyr Phe Thr Asn Glu Met Lys Gin
35 40 45

Leu Gin Asp Asn Tyr Ser Lys lie Gly Leu Ala Cys Phe Ser Lys Thr
50 55 60

Asp Glu Asp Phe Trp Asp Ser Thr Asp Gly Glu lie Leu Asn Arg Pro
65 70 75 80

Cys Leu Gin Val Lys Arg Gin Leu Tyr Gin Leu lie Glu Glu Val Thr

85 90 95

Leu Arg Thr Phe Gin Asp Thr lie Ser Thr Val Pro Glu Lys Gin Leu

100 105 110

Ser Thr Pro Pro Leu Pro Arg Gly Gly Arg Pro Gin Lys Val Ala Ala
115 120 125

His lie Thr Gly lie Thr Arg Arg Ser Asn Ser Ala Leu lie Pro lie
130 135 140

Ser Lys Asp Gly Lys Thr Leu Gly Gin Lys lie Glu Ser Trp Glu Ser
145 150 155 160

Ser Arg Lys Gly His Ser Phe Leu Asn His Val Leu Phe Arg Asn Gly

165 170 175

Glu Leu Val lie Glu Gin Glu Gly Leu Tyr Tyr lie Tyr Ser Gin Thr

180 185 190

Tyr Phe Arg Phe Gin Glu Ala Glu Asp Ala Ser Lys Met Val Ser Lys
195 200 205

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Asp Lys Val Arg Thr Lys Gin Leu Val Gin Tyr lie Tyr Lys Tyr Thr
210 215 220

Ser Tyr Pro Asp Pro lie Val Leu Met Lys Ser Ala Arg Asn Ser Cys
225 230 235 240

Trp Ser Arg Asp Ala Glu Tyr Gly Leu Tyr Ser lie Tyr Gin Gly Gly

245 250 255

Leu Phe Glu Leu Lys Lys Asn Asp Arg lie Phe Val Ser Val Thr Asn

260 265 270

Glu His Leu Met Asp Leu Asp Gin Glu Ala Ser Phe Phe Gly Ala Phe
275 280 285

Leu lie Asn
290

(2) INFORMATION FOR SEQ ID NO: 7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 8 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY : not relevant

(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE : NO

(vii) IMMEDIATE SOURCE:

(B) CLONE: FLAG peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:

Asp Tyr Lys Asp Asp Asp Asp Lys
1 5

(2) INFORMATION FOR SEQ ID NO: 8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: not relevant

(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO

(vii) IMMEDIATE SOURCE:

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(B) CLONE: conserved peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:

Leu Val Val Xaa Xaa Xaa Gly Leu Tyr Tyr Val Tyr Xaa Gin Val Xaa
15 10 15

•i

Phe

(2) INFORMATION FOR SEQ ID NO: 9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 32 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: not relevant

(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO

(vii) IMMEDIATE SOURCE:

(B) CLONE: CMV leader

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 9 :

Met Ala Arg Arg Leu Trp lie Leu Ser Leu Leu Ala Val Thr Leu Thr
1 5 10 15

Val Ala Leu Ala Ala Pro Ser Gin Lys Ser Lys Arg Arg Thr Ser Ser

20 25 30

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PCT/US96/10895

CLAIMS

What is claimed is:

L An isolated DNA encoding a TRAIL polypeptide, wherein said TRAIL polypeptide
comprises an amino acid sequence that is at least 80% identical to an amino acid
sequence selected from the group consisting of amino acids 1 to 281 of SEQ ID NO:2
and amino acids 1 to 291 of SEQ ID NO:6, wherein said TRAIL polypeptide is capable
of inducing apoptosis of Jurkat cells.

2. An isolated DNA of claim 1, wherein said TRAIL polypeptide comprises an amino
acid sequence selected from the group consisting of amino acids 1 to 28 1 of SEQ ID
NO:2 and amino acids 1 to 291 of SEQ ID NO:6.

3. An isolated DNA encoding a soluble TRAIL polypeptide, wherein said soluble
TRAIL polypeptide comprises an amino acid sequence that is at least 80% identical to a
sequence selected from the group consisting of:

a) the extracellular domain of human TRAIL (amino acids 39 to 28 1 of SEQ ID
NO:2); and

b) a fragment of said extracellular domain;

wherein said soluble TRAIL polypeptide is capable of inducing apoptosis of Jurkat
cells.

4. A DNA of claim 3, wherein said soluble TRAIL polypeptide comprises an amino
acid sequence selected from the group consisting of:

a) the extracellular domain of human TRAIL (amino acids 39 to 28 1 of SEQ ID
NO:2); and

b) a fragment of said extracellular domain, wherein said fragment is capable of
inducing apoptosis of Jurkat cells.

5. A DNA of claim 4, wherein said soluble TRAIL polypeptide comprises the
sequence of amino acids x to 281 of SEQ ID NO:2, wherein x represents an integer
from 39 to 95.

6. A DNA of claim 5, wherein said soluble TRAIL polypeptide comprises the
sequence of amino acids 95 to 28 1 of SEQ ID NO:2.

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PCT7US96/10895

7. A DNA of claim 3, wherein said soluble TRAIL polypeptide comprises conservative
substitution(s) in an amino acid sequence selected from the group consisting of:

a) the extracellular domain of human TRAIL (amino acids 39 to 28 1 of SEQ ID
NO:2); and

b) a fragment of said extracellular domain;

wherein the conservatively substituted TRAIL is capable of inducing apoptosis of
Jurkat cells.

8. An expression vector comprising a DNA according to any one of claims 1 to 7.

9. A process for preparing a TRAIL polypeptide, comprising culturing a host cell
transformed with a vector according to claim 8 under conditions promoting expression
of TRAIL, and recovering the TRAIL polypeptide.

10. A purified TRAIL polypeptide comprising an amino acid sequence that is at least
80% identical to an amino acid sequence selected from the group consisting of amino
acids 1 to 281 of SEQ ID NO:2 and amino acids 1 to 291 of SEQ ID NO:6, wherein
said TRAIL polypeptide is capable of inducing apoptosis of Jurkat cells.

1 1. A purified TRAIL polypeptide of claim 10, comprising an amino acid sequence
selected from the group consisting of amino acids 1 to 281 of SEQ ID NO:2 and amino
acids 1 to 291 of SEQ ID NO:6.

12. A human TRAIL polypeptide encoded by the cDNA insert of the recombinant
vector deposited in strain ATCC 69849.

13. A purified soluble TRAIL polypeptide comprising an amino acid sequence that is at
least 80% identical to a sequence selected from the group consisting of:

a) the extracellular domain of human TRAIL (amino acids 39 to 28 1 of SEQ ID
NO:2); and

b) a fragment of said extracellular domain;

wherein said soluble human TRAIL polypeptide is capable of inducing apoptosis of
Jurkat cells.

14. A TRAIL polypeptide of claim 13, comprising an amino acid sequence selected
from the group consisting of:

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PCTYUS96/10895

a) the extracellular domain of human TRAIL (amino acids 39 to 28 1 of SEQ ID
NO:2); and

b) a fragment of said extracellular domain, wherein said fragment is capable of
inducing apoptosis of Jurkat cells.

15. A TRAIL polypeptide of claim 14, comprising the sequence of amino acids x to
281 of SEQ ID NO: 2, wherein x represents an integer from 39 to 95.

16. A TRAIL polypeptide of claim 15, comprising amino acids 95 to 281 of SEQ ID
NO:2.

17. A TRAIL polypeptide of claim 13, wherein said soluble TRAIL polypeptide
comprises conservative substitution(s) in an amino acid sequence selected from the
group consisting of:

a) the extracellular domain of human TRAIL (amino acids 39 to 28 1 of SEQ ID
NO:2); and

b) a fragment of said extracellular domain;

wherein the conservatively substituted TRAIL is capable of inducing apoptosis of
Jurkat cells.

18. An oligomer comprising from two to three soluble TRAIL polypeptides of claim
13.

19. A TRAIL trimer comprising three soluble TRAIL polypeptides of claim 16.

20. An antibody that specifically binds a TRAIL protein of claim 1 1 or 15.

21. An antibody according to claim 20, wherein said antibody is a monoclonal
antibody.

WO 97/01633

PCT/US96/10895

2/2

+ + +

+ + + + +

+ 4- + +

+ +

+ + +

•2

• ■ ■

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CD CD CD

INTERNATIONAL SEARCH REPORT

International application No.
PCT/US96/10895

A. CLASSIFICATION OF SUBJECT MATTER
IPC(6) : Please See Extra Sheet.

US CL :536/23.5; 435/69.1, 252.3, 320.1; 530/350, 387.9, 388.1
According to International Patent Classification (IPC) or to both national classification and IPC

B. FIELDS SEARCHED

Minimum documentation searched (classification system followed by classification symbols)
U.S. : 536/23.5; 435/69.1, 252.3, 320.1; 530/350, 387.9, 388.1

Documentation searched other than minimum documentation to the extent that such documents are included in the fields searched

Electronic data base consulted during the international search (name of data base and, where practicable, search terms used)
Please See Extra Sheet.

C. DOCUMENTS CONSIDERED TO BE RELEVANT

Category*

Citation of document, with indication, where appropriate, of the relevant passages

Relevant to claim No.

A,P

O'MAHONEY, A.M. An Immune Supressive Factor Derived
from Esophageal Squamous Carcinoma Induces Apoptosis in
Normal and Transformed Cells of Lymphoid Lineage. The
Journal of Immunology. 01 November 1993, Vol.151, No. 9,
pages 4847-4856.

US 5,512,435 A (RENSCHLER ET AL) 30 April 1996.

1-21

I | Further documents are listed in the continuation of Box C. | | See patent family annex.

■A"

•E*
"L"

Special categories of cited documents:

document defining the general stale of the art which is not considered
to be of particular relevance

earlier document published on or after the international filing date

document which may throw doubts on priority claim(s) or which is
cited to establish the publication date of another citation or other
special reason (as specified)

document referring to an oral disclosure, use, exhibition or other

•x-

•Y"

document published prior to the international filing date but later than
the priority date claimed

later document published after the international filing date or priority
date and not in conflict with the application but cited to understand the
principle or theory underlying the invention

document of particular relevance; the claimed invention cannot be
considered novel or cannot be considered to involve an inventive step
when the document is taken alone

document of particular relevance; the claimed invention cannot be
considered to involve an inventive step when the document is
combined with one or more other such documents, such combination
being obvious to a person skilled in the art

document member of the same patent family

Date of the actual completion of the international search
14 AUGUST 1996

Date of mailing of the international search report

16SEP1996

Name and mailing address of the ISA/US
Commissioner of Patents and Trademarks
Box PCT

Washington, D.C. 20231
Facsimile No. (703) 305-3230

Authorized officer _ * ^_ »

DARYL A. BASHAM « V^V^^
Telephone No. (703) 305-0196

Form PCT/ISA/210 (second sheet)(July 1992)*

INTERNATIONAL SEARCH REPORT

International application No.
PCT/US96/10895

Box I Observations where certain claims were found unsearchable (Continuation of item 1 of first sheet)

This international report has not been established in respect of certain claims under Article 17(2)(a) for the following reasons:

1. |"™| Claims Nos.:

because they relate to subject matter not required to be searched by this Authority, namely:

□

Claims Nos.:

because they relate to parts of the international application that do not comply with the prescribed requirements to such
an extent that no meaningful international search can be carried out, specifically:

3. | | Claims Nos.:

because they are dependent claims and are not drafted in accordance with the second and third sentences of Rule 6.4(a).

Box II Observations where unity of invention is lacking (Continuation of item 2 of first sheet)
This International Searching Authority found multiple inventions in this international application, as follows:
Please See Extra Sheet.

1 • [x] As all required additional search fees were timely paid by the applicant, this international search report covers all searchable
claims.

I — I

| | As all searchable claims could be searched without effort justifying an additional fee, this Authority did not invite payment

of any additional fee.

As only some of the required additional search fees were timely paid by the applicant, this international search report covers
only those claims for which fees were paid, specifically claims Nos.:

4 ' O N ° rec l uired add itional search fees were timely paid by the applicant. Consequently, this international search report is
restricted to the invention first mentioned in the claims; it is covered by claims Nos.:

Remark on Protest

I — I

J | The additional search fees were accompanied by the applicant's protest.

I 1 No protest accompanied the payment of additional search fees.

Form PCT/ISA/210 (continuation of first sheet(l))(July 1992)*

INTERNATIONAL SEARCH REPORT

International application No.
PCT/US96/10895

A. CLASSIFICATION OF SUBJECT MATTER:
IPC (6):

C12N 15/09, 15/17, 15/63; C07H 21/04; C07K 1/14, 14/52, 16/24; C12P 1/20, 21/08

B. FIELDS SEARCHED

Electronic data bases consulted (Name of data base and where practicable terms used):
DIALOG, DERWENT, MEDLINE, APS, BIOSIS, SCISEARCH, STN

search terms: FAS ligand, apoptosis ligand, jurkat clone, cells, TNF-related, apoptosis inducing, regulating, soluble
FAS, T-cell leukemia, angiogenesis, CD2

BOX II. OBSERVATIONS WHERE UNITY OF INVENTION WAS LACKING
This ISA found multiple inventions as follows:

I. Claims 1-19, drawn to isolated DNA, a vector, a process for preparing a polypeptide, a host cell and an oligomer.

II. Claims 20 and 21, drawn to antibodies.

and it considers that the International Application does not comply with the requirements of unity of invention (Rules
13.1, 13.2 and 13.3) for the reasons indicated below:

The DNA and the polypeptide compositions of Group I have materially different structures and biological functions
from the antibodies of Group II. The special technical features by which the DNA and polypeptide of Group I are
defined distinguish them from the special technical features which define the antibodies of Group II. Because special
technical features are not shared between Groups I and II, unity of invention is lacking. The claims are not so linked by
a special technical feature within the meaning of the PCT Rule 13.2 so as to form a single inventive concept.

Form PCT/ISA/210 (extra sheet)(July 1992)*

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